ACCESSORY FACTOR FUNCTION FOR INTERFERON GAMMA AND ITS RECEPTOR

Abstract: This invention relates (a) to a 540 kb YAC which encodes the necessary species-specific factor(s) and is able to substitute for human Chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens; (b) to the construction of a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs in order to facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones; (c) to two fragmentation vectors, pSE1 and pSE2, which contain a neomycin resistance and URA3 gene, developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA; (d) to a chromosomal fragmentation procedure employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC (GART D142H8) known to map to Chromosome 21q and to encode the human interferon-gamma receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene; and (e) to the isolation of cDNA clones that encode the necessary species-specific factor and that are able to substitute for human Chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens.

Description: 5 f 10 ACCESSORY FACTOR FUNCTION FOR INTERFERON GAMMA AND ITS RECEPTOR 15 Field Of The Invention This invention relates (a) to a 540 kb YAC which encodes the necessary species-specific factor(s) and is able to substitute for human 20 Chromosome 21 to reconstitute the Bu-JFN-gamma receptor-mediated induction of class I HLA antigens; (b) to the construction of a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs in order to facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones; (c) to two 25 fragmentation vectors, pSEl and pSE2, which contain a neomycin resistance and URA3 gene, developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA; (d) to a chromosomal fragmentation procedure employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC (GART D142H8) known to map to Chromosome 21q and to encode 30 the human interferon-g-wnmα receptor (Hu-ΪFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene; and (e) to the isolation of cDNA clones that encode the necessary species-specific factor and that are able to substitute for human Chromosome 21 to reconstitute the Hu- ΪFN-gamma receptor-mediated induction of class I HLA antigens. 35 Background Of The Invention The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography. YAC Clone Encoding an Accessory Factor Human interferon gamma (Hu-IFN-gamma) induces a variety of biological responses such as antiviral, antiproliferative, and immunomodulatory activities in sensitive cells (1-5). Immunoregulatory functions induced by Hu-IFN- gamma such as induction of class I and class II human HLA antigens, activation of macrophages, regulation of Ig class switching, and up-regulation of Fc receptor expression are involved in modulating a variety of other host defense mechanisms (5-10). The first event in inducing these responses is the specific binding of IFN- gamma to its cell surface receptor encoded on human Chromosome 6 (11) or mouse Chromosome 10 (12,64). However, human Chromosomes 6 and 21 (14,15) and mouse Chromosomes 10 and 16 (16) are required for sensitivity to IFN-gamma as measured by the induction of class I MHC antigens, which indicates that the binding of IFN-gamma to the receptor is necessary, but not sufficient to induce these antigens. Interaction between the extracellular domain of the Hu-IFN-gamma receptor and the species-specific accessory factor (AF-1) which leads to class I HLA antigen induction upon treatment with Hu-IFN-gamma was suggested by experiments with chimeric receptors (17-19). A five-amino acid sequence (YDKPH) of the intracellular domain of the Hu-IFN-gamma receptor is required for this activity (20,21). Moreover, at least two additional accessory factors (AF-2 and AF-3) are involved in EMCV and VSV antiviral activity (20,65). However, little is known about how the binding of Hu-IFN-^omm to its receptor initiates this diverse array of functional changes and the nature of the accessory factors. The region of Chromosome 21 which is necessary for sensitivity to Hu-IFN-gamma as assayed by the expression of class I HLA antigens was localized within a region of about 1-3 mb of Chromosome 21q with irradiation-reduced hamster/human somatic hybrid cells (22). The cloned Hu-IFN-gamma receptor cDNA (23,24) was expressed in hamster/human hybrid cells which have human Chromosome 21q as the sole human Chromosome to reconstitute a biologically- active Hu-IFN-gamma receptor for HLA class I antigen induction (25), and EMCV antiviral protection activity (20). Thus, it appeared that human Chromosome 21q encodes all necessary factors for both activities. This was confirmed by another study with mouse/human hybrid cells transfected with the Hu-IFN-gamma receptor cDNA that showed that human Chromosome 21 is sufficient for induction of class I MHC antigens, EMCV antiviral activity and induction of 2',5'-oligoadenylate synthetase in response to Hu-IFN-gamma (26). The cloning of DNA into yeast artificial chromosomes (YACs) has allowed isolation of much larger DNA fragments than was previously possible (27). Also, development of polymerase chain reaction (PCR)-based YAC screening methods has facilitated the identification of specific YAC clones (28). These large cloned segments have potential applications for the isolation of functional domains from chromosomes (29). To assay for the biological functions of genes included in YACs, yeast cells can be fused to cultured mammalian cells. Experiments to demonstrate the functional expression of genes carried on YACs have been carried out with G6PD (glucose-6-phosphate dehydrogenase), HPRT (hypoxanthine phosphoribosyltransferase) and GART YAC clones (30-32). To allow selection for fused cells in these cases, a gene cassette that confers resistance to antibiotic G418 on mammalian cells can be introduced into the YAC by homologous recombination targeted to human genomic sequences (33,34). ΛZα-targeting YAC Deletion Plasmids Yeast artificial chromosomes (YACs) provide a system for the cloning of up to one megabase of contiguous DNA (55,56). While the large size of the YAC insert facilitates the isolation of specific large segments of DNA (59), identification and localization of genes within the YAC requires conventional yeast genetic techniques to manipulate the YAC insert (39). Chromosome fragmentation has been developed for Saccharomyces cerevisiae YPH252 with a fragmentation plasmid (48,60). Fragmentation takes advantage of homologous recombination between Alu family sequences or LINE human repetitive DNA elements in the fragmentation plasmid and the YAC insert. The recombination event causes chromosome breakage at the site where homologous integration occurs and deletion of all portions of the YAC distal to the targeted site. Similarly another fragmentation plasmid system has been developed for S. cerevisiae AB1380 (58). The plasmid developed by Cook et al. contained the gene for neomycin phosphotransferase as a eukaryotic selectable marker. A second type of YAC modification plasmid, an integrating plasmid, has been described (33) for S. cerevisiae YPH252. In contrast to the fragmentation plasmid, the integrating plasmid contains two recombinogenic sequences resulting in the insertion of the plasmid into the YAC. The integrating plasmid has been used mainly to insert a neomycin-resistance cassette to be used as a selectable marker following fusion of YAC-containing yeast spheroplasts with mammalian cells (33). Another integrating plasmid containing a DNA fragment derived from the DNA of the YAC insert, not Alu family sequences, has been described (34). Yeast Artificial Chromosome Fragmentation Vectors Yeast artificial chromosome vectors have enabled the cloning of up to 1 megabase of DNA (27). In order to reduce YACs to smaller sizes for mapping and manipulation, Reeves et al. (62) and Pavan et al. (48) have developed fragmentation vectors which are capable of producing a set of deletions in a parental YAC containing human genomic DNA. The fragmented YACs obtained by this procedure are useful not only for mapping studies but also for determining the location of genes encoding certain biochemical functions. The vectors which have been reported utilize transformation to a His phenotype to select for clones containing fragmented YACs (48,60). While this procedure is applicable to YACs carried in his3 " YPH252 yeast cells, it cannot be used with the AB1380 yeast strain which is the host for many YAC libraries. Localization of the Human Interferon-gαmm Receptor Accessory Factor Gene The human and murine interferon gamma receptors (IFN-gamma R) have been shown to be homologous to a considerable degree (94). Nevertheless, these receptors are very specific in terms of binding human and murine IFN-gamma, respectively (95). A second level of species specificity involves the IFN-gamma accessory factor. The existence of the IFN-gamma accessory factor was first postulated because experiments with somatic cells hybrids indicated that the chromosome encoding the gene for the human IFN-gamma R (Chromosome 6) is itself not sufficient for biological activity even though human IFN-gamma can bind to the receptor (96-98). Only when human Chromosome 6 is complemented with human Chromosome 21 in somatic cell hamster x human hybrids does Hu-IFN- gamma signal transduction occur (97.98). Similarly, only when murine Chromosome 10 encoding the Mu-IFN-χamma R (99) is accompanied by murine Chromosome 16 (encoding the murine accessory factor) does the Mu-IFN-gamma R become biologically active in hamster x murine somatic cell hybrids (100). The human Chromosome 21 accessory factor is likely to be a membrane- associated protein as several studies have shown that the accessory factor and the extracellular domain of the IFN-gamma R must be from the same species for signal transduction to occur (101-103). Although interactions with the transmembrane and intracellular domains have not been ruled out, these interactions, if they exist, are not species specific. Use of radiation-reduced somatic cell hybrids has enabled further mapping of the accessory factor gene on Chromosome 21 (104). To date, the most accurate estimate of the chromosomal location of the accessory factor gene as determined in somatic cell hybrids indicates that the gene is located proximal to marker D21S58 (104). More recent data from this laboratory indicate that accessory factor activity is encoded by the yeast artificial chromosome (YAC) whose address on chromosome 21 is D142H8 (105). This YAC, designated GART D142H8 because it also encodes the GART gene, is 540 kb in length and is located proximal to the YAC which encodes the human IFN-α/B receptor (105). Throughout the specification and claims, the following abbreviations will stand for the words and phrases set out below. A, Ala; C, Cys; D, Asp; E, Glu; F, Phe, G, Gly; H, His; I, He; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. 2,5-A π : (2'-5')-oligoadenylate: (2'-5')-oligo(adenylic) acid; oligoadenylic acid with 2', 5'-phosphodiester linkages: also abbreviated as (2'-5')-oligio(A), pppA(2'p5'A) π , pppA(2'-5')A rt , or (2'-5')p3A3; 2-5A is used as well as 2,5-p3A . ^260 umt: The quantity of material that yields an absorbance of 1.0 when measured at 260nm in a cuvette with a path length of 1.0 cm. Analogously, AgQO represents measurements at 600 nm, A550 at 550 nm, etc. This quantity of material is also sometimes designated as OD260 > OD550, O D 600 > etc - Act D: Actinomycin D BES: N,N-bis[2-Hydroxyl]-2-aminoethanesulfonic acid; 2-[bis(2- hydroxethyl)amino]-ethanesulfonic acid BP: Base pairs; usually used as lower case bp. Bq: Becquerel; 1 Bq = 1 dps or 60dpm; 1 Ci = 3.7 x 10 10 Bg; TBq = 10 12 Bq BSA: Bovine serum albumin Con A: Concanavalin A CPD: Citrate phosphate dextrose solution CPE: Cytopathic effect DEAE Cellulose: Diethylaminoethylcellulose DHFR: Dihydrofolate reductase DNA: Deoxyribonucleic acid cDNA: Complementary DNA dsDNA: Double-stranded DNA DRB: 5,6-Dichloro-l-bet -D-ribofuranosylbenzimidazole dsRNA: Double-stranded RNA DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid EGTA: Ethylene glycol bis(t etα-aminoethyl ether)-N, N'-tetraacetic acid EID50: Egg infectious dose; concentration at which half the eggs are infected. EMCV: Encephalomyocarditis virus EMEM: Eagle's minimal essential medium ESS: Earle's salt solution FBS: Fetal bovine serum FCS: Fetal calf serum GPT: Guanosine phosphoribosyltransferase (recommended name is guanosine phosphorylase; systematic name is guanosine: orthophosphate ribosyltransferase HBSS: Hanks' balanced salt solution HEPES: N-2-Hydroxethylpiperazine-N'-2-ethanesulfonic acid IU: Units of interferon with respect to the appropriate international reference standard. KBP: Kilobase pairs; usually used as lower case kbp. MDMP: 2-(4-Methyl-2, 6-dinitroanilino)-N-methyl-propionamide MEM: Minimal essential medium (EMEM, unless otherwise noted). Mg(OAc)2: Magnesium acetate MOI: Multiplicity of infection NAD: Nicotinamide adenine dinucleotide NaOAc: Sodium acetate Natural interferon: This term refers to interferon produced by animal cells after induction in contrast to interferon produced by recombinant DNA technology. In some cases the two (i.e. , the interferon produced by animal cells and the same species by recombinant DNA technology) may be chemically identical. NDV: Newcastle disease virus PBL: Peripheral blood leukocytes PBMC: Peripheral blood mononuclear cells PBS: Phosphate-buffered saline PFC: Plaque-forming cell PFU: Plaque-forming unit PHA: Phytohemagglutinin Poly(I) . poly(C): Polyinosinic acid . polycytidylic acid, double stranded synthetic homopolymers [alternatively poly(rl) . poly(rC)] RNA: Ribonucleic acid dsRNA: double-stranded RNA SDS: Sodium dodecyl sulfate SDS-Page: SDS-Polyacrylamide gel electrophoresis SEA: Staphylococcal enterotoxin A SEB: Staphylococcal enterotoxin B SSC: Standard saline citrate solution, 0.15 M NaCl/0.015 M sodium citrate, adjusted to pH 7.0 with NaOH. TCA: Trichloroacetic acid TCID50: Tissue culture infectious dose; concentration at which half the cultures are infected. TEMED: N,N,N' ,N'-Tetramethylethylenediamine TES: N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Tricine: N-Tris(hydroxymethyl)methylglycine TPA: The phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Tris: Tris(hydroxymethyl)aminomethane VSV: Vesicular stomatitis virus Other Abbreviations: YAC, yeast artificial chromosome; PFGE, pulsed-field gel electrophoresis; Hu-IFN-gamma, human interferon gamma; HLA, human leucocyte antigen; CHO, Chinese hamster ovary; GART, phosphoribosylglycinamide formyltransferase; EMCV, encephalomyocarditis virus; VSV, vesicular stomatitis virus. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a pulse field gel electrophoresis of various YACs and hybridization to total human DNA. Figure 2 illustrates a Southern hybridization. Figure 2A illustrates the agarose plugs from neo τ Lys2+ transformants derived from each YAC clone were analyzed by PFGE and the blot was probed with the labelled neo τ gene to determine whether or not the neo r gene was targeted to the YAC. Figure 2B shows that yeast chromosomal DNAs from the same YAC clones as in A were digested with BamHI and Xho to release the neo τ gene cassette and the blot was probed with the labelled neo τ gene. Figure 3 shows the induction of HLA-B7 surface antigen in 16-9 cells fused with the YAC-JS2. Figure 4 illustrates the integration/deletion plasmid and its use. The schematic diagram shows two- point recombination between the integration/deletion plasmid and YAC insert that results in internal deletions. Figure 5 illustrates a restriction map of the integration/deletion plasmid. The plasmid contains the antibiotic G418 resistance gene cassette under control of the thymidine kinase promoter and the LYS2 gene as a yeast selectable marker. Figure 6 illustrates a Southern blot of PFGE from the deleted YACs. Figure 7 illustrates the localization of integrated plasmid in targeted YAC. Figure 8 illustrates the use of deleted YAC to identify specific genes and cDNA clones. Figure 9 illustrates an agarose gel electrophoresis of DNA and Southern blot analysis. Figure 10 illustrates a schematic map of fragmentation vectors pSEl and pSE2, and the procedure used for fragmentation of the parental GARTD142H8.ura.2 YAC. TEL = telomere; CEN = centromere. Figure 11 shows a Southern blot of YACs to demonstrate the presence of the unique pGC8.10E6 sequence. Figure 12 illustrates YAC J 18 and overlapping cosmids. Figure 13 shows a Northern blot analysis of RNA isolated from human Raji, HL60 and 3xlS cells. (A) Three micrograms of oligo(dT)-selected mRNA from 3xlS cells was fractionated on a 1.0% agarose gel containing 2.2 M formaldehyde and blotted onto a nylon membrane. (B) About one half microgram of oligo(dT)-selected mRNA from Raji and HL60 cells were processed as described above and probed with a 1.8 kb insert from plasmid pJS3. Figure 14 is a schematic representation and restriction map of the human IFN-gamma Accessory Factor-1 (AF-1) cDNA clones pSKl and pJS3. Figure 15 A illustrates human IFN-gamma accessory factor- 1 (AF-1) cDNA (plasmid pSKl) nucleotide and predicted amino acid sequences. Figure 15B illustrates the cDNA sequence of the insert in plasmid pJS3, another clone with accessory factor function. Figure 16 is a hydropathy plot of the predicted human IFN-gamma accessory factor- 1 amino acid sequence. Figure 17 shows the induction of HLA-B7 surface antigen. Figure 18 shows the amino acid sequence alignment of human AF-1 and some members of the class 2 cytokine receptor family. Figure 19 shows the accessory factor and interferon α/β receptor region of Chromosome 21q. Figure 20 is a schematic illustration of YACs used to transform 16-9 cells. Figure 21 is a cytofluorographic analysis of MHC class I induction in cells transformed by yeast containing Ura+ YACs. Figure 22 shows the responsiveness of cell lines to various concentrations of Hu-IFN-gαmmα. Figure 23 is a gel shift assay for ISGF3-gαmmβ(A) and GAF(B) in selected cell lines. Figure 24 is a diagrammatic representation of the location and interaction of the Hu-IFN-gamma receptor accessory factors located on Chromosome 21q and elsewhere. SUMMARY OF THE INVENTION This invention relates (a) to a 540 kb YAC which encodes the necessary species-specific factor(s) and is able to substitute for human Chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens; (b) to the construction of a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs in order to facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones; (c) to two fragmentation vectors, pSEl and pSE2, which contain a neomycin resistance and URA3 gene, developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA; (d) to a chromosomal fragmentation procedure employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC (GART D142H8) known to map to Chromosome 21q and to encode the human interferon-gα/røwfl receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene; and (e) to the isolation of cDNA clones that encode the necessary species-specific factor and that are able to substitute for human Chromosome 21 to reconstitute the Hu- IFN-gamma receptor-mediated induction of class I HLA antigens. DETAILED DESCRIPTION OF THE INVENTION YAC Clone Encoding an Accessory Factor Human Chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma) as measured by the induction of human HLA class I antigen. Whereas human Chromosome 6 encodes the receptor for Hu-IFN-gamma, human Chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human Chromosome 21 which is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation- reduced fragment of human Chromosome 21. The cell line with the minimum Chromosome 21-specific DNA is CHO 3xlS. In order to localize the genes further, 10 different YAC (yeast artificial chromosome) clones from 6 different loci in the vicinity of the 3xlS region were fused to a human x hamster hybrid cell line (designated 16-9) which contains human Chromosome 6q (supplying the Hu-IFN- gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. It was found that a 540 kb YAC encodes the necessary species-specific factor(s) and is able to substitute for human Chromosome 21 to reconstitute the Hu-IFN-gαmmα receptor-mediated induction of class I HLA antigens. However, the factor encoded on the YAC does not confer antiviral protection against encephalomyocarditis virus demonstrating that an additional factor encoded on human Chromosome 21 is required for the antiviral activity. A α-targeting YAC Deletion Plasmids In order to facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones, a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs was constructed. The linearized integration/deletion plasmid, which contains Alu family sequences at both ends, can recombine with YACs containing human repetitive sequences via homologous recombination. The homologous recombination results in a random integration of the antibiotic G418 resistant gene into a human genomic Alu sequence, and in most cases, an internal deletion within the YAC. The YACs with internal deletions can be useful to identify the location of the genes if they produce functional knockouts. In those cases when the integration/deletion event disrupts the integrity of the gene so it no longer can produce a viable and functional mRNA in fused eukaryotic cells, the site of integration in the YAC thus serves as a marker for the inactivated gene. In this report we describe a new YAC integration/deletion plasmid for S. cerevisiae AB1380 to locate specific genes in YACs. This plasmid contains a human Alu family sequence (BLUR 13) arranged in such a way that the plasmid integrates following two homologous recombination events within the YAC insert. These events result in the integration of the linearized plasmid at two Alu sequences resulting in the deletion of sequences between the sites of recombination (Figure 4). Yeast Artificial Chromosome Fragmentation Vectors Two fragmentation vectors, pSEl and pSE2, were developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA. Ura " yeast cells containing YACs with a URA3 marker were selected with 5-fluoro- orotic acid. Fragmented YACs were subsequently generated by transformation to a Ura + phenotype. Over eighty percent of the transformants contained YACs of reduced molecular size. These fragmented YACs will prove to be useful in mapping the region of human chromosomes covered by the parental YAC. Fragmentation utilizing URA3 transformation provides a method for producing YAC deletion sets from YACs contained in AB1 80 and other Ura3 " yeast stains. Linkage of a neomycin resistance gene to the URA3 gene facilitates functional analysis of these YACs in eukaryotic cells. Localization of the Human Interferon-gαmmα Receptor Accessory Factor Gene A chromosomal fragmentation procedure was employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC (GART D142H8) known to map to Chromosome 21q and to encode the human interferon- gamma receptor (Hu-IFN-gαmmc R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene. Activity of the accessory factor gene in cell lines produced from the fusion of fragmented, neo YACs to CHO cells expressing the Hu-IFN-gamma R was used to localize the accessory factor gene. These deleted YACs retain accessory factor activity in Chinese hamster ovary cells until the deletions from the acentric end exceed 390 kb. Therefore, the accessory factor (AF-1) gene can be localized to a 150 kb region at the left (centric) end of the parental 540 kb GART YAC. Cell lines produced from the fusion of fragmented, neo + YACs to CHO cells with the Hu-IFN-gαmmα R stably express this accessory factor. Cells containing functional YACs, as judged by MHC inducibility by IFN-gamma, are also able to induce the ISGF3-gαmmα and GAF transcription factors. However, cell lines containing the complete AF-1 gene were not protected against encephalomyocarditis virus (EMCV) by Hu-IFN-gαmmc although antiviral protection induced by IFN-gamma remained intact. Other CHO cell lines containing the Hu-IFN-gamma R and human Chromosome 21q have been shown to be protected against EMC, but not vesiculostomatitis virus (VSV) by Hu- ΪFN-gamma. Therefore, the Hu-IFN-gamma R and the accessory factor are sufficient for some, but not all, of the actions of the Hu-IFN-gommα. We postulate that an additional accessory factor required for antiviral activity is encoded on Chromosome 21q; and that at least two additional accessory factors are required for generation of antiviral activity against both EMCV and VSV. cDNA Clone Encoding an Accessory Factor The gene for one of the accessory factors was localized to a small genomic region (Soh et al. , Proc. Natl. Acad. Sci. , in press) through the identification of functional YAC clones. With the use of probes isolated from the genomic clone, we identified cDNA clones expressing the accessory factor. Here we report isolation of cDNA clones that encode the necessary species-specific factor and that are able to substitute for human Chromosome 21 to reconstitute the Hu- IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer antiviral protection against encephalomyocarditis virus confirming that an additional factor encoded on human Chromosome 21 is required for reconstitution of antiviral activity; and that a family of such accessory factors responsible for different actions of IFN-gamma exist. As set out above, the present invention pertains to the Accessory Factor-1 (AF-1) for Human Interferon Gamma Function, each of the Variants of AF-1, and all other accessory factors or other factors isolated with the use of AF-1 constructs, transfected cells, etc., to isolate and define the factors. The present invention also pertains to antibodies to AF-1 , such as monoclonal and polyclonal antibodies to AF-1. Antibodies to AF-1 may be employed (a) for the diagnosis of AF-1 deficiencies and excesses; (b) for tumor diagnosis (AF-1 in serum and urine); (c) for blocking of AF-1 specific mediated functions of IFN-gαmmα; and (d) for the treatment of diseases where soluble IFN-gamma receptors or IFN-gamma immunoadhesins are useful to block function of IFN-gαmmα. The present invention further pertains to soluble AF-1 which may be employed (a) for use a as standard in diagnosis as set out above ; (b) for blocking the activity of the IFN-gαmmα receptor; and (c) for activating the AF-1 receptor with appropriate constructs of AF-1. The present invention also pertains to (a) the use of AF-1 in gene therapy to reconstitute a defective IFN-gamma (systems defective in AF-1); (b) the use of oligonucleotides from the AF-1 sequence for diagnosis of AF-1 defects in tumors and other diseases; (c) the use of the nucleotide sequence (DNA, RNA) of AF-1 and its genes to use for diagnosis of tumors, other diseases, and defects in AF-1 function and expression; (d) the use of the AF-1 probes to isolate animal correlates of AF-1 so that animal AF-1 sequences will be covered (cat, bovine, dog, horse, mouse, rat, monkey, hamster, rabbit, goat, sheep, all mammals); (e) the nucleotide sequence of AF-1 and all closely related sequences that hybridize to the probes designed from the AF-1 sequence; (f) transgenic mice (animals) containing human AF-1 and the human IFN-gamma receptor of chimeric human/mouse IFN-gamma receptors to construct transgenic animals with a functional human IFN-gamma system (this will allow testing of the species specific IFN-gamma in mice for side effects and efficacy. At the present time, only species-specific cytokines as IFN-gamma can only be tested in humans. This can revolutionize the testing of such human cytokines in animal models); and (f) plasmids designed for Manipulation of YACs. Use of AF-1 to Isolate Other Accessory Factors: The identification of AF-1 and the discovery of at least two other accessory factors provides a methodology for isolation of the other accessory factors and determining if there are more members of this family. The use of AF-1 together with other cosmids, YACs, or cDNA clones representing the 3xlS region of Chromosome 21 will permit a determination of whether AF-2 (the accessory factor required for EMCV protection by Hu-IFN-gαmmα) can function alone or requires AF-1. Furthermore, assay of other activities induced by IFN-gamma can be measured to define whether these are activities mediate through AF-1: these include antiproliferative activity, stimulation of T-cell functions, stimulation of macrophage (and similar cells) functions, other antiviral activities, stimulation of natural killer cells, etc. AF-1 in Therapy of Cancer, Modulation of Class I Major Histocompatibility (MHC) Antigen Expression on Cells Because the IFN-gamma system is one of the most potent general growth regulators of cells and is also a powerful modulator of the immune system, there will be many uses for these accessory factors. These observations imply that the interferon system in general, and IFN-gamma specifically, is effective tumor suppression through two different avenues: direct action on tumor cells; and modulation of the immune system. Modifications in the level of AF-1 in tumor cells can be used as diagnostic criteria for cancer and prognosis; replacement of missing functions through gene therapy of tumor cells or other somatic cells such as lymphocytes and macrophages. Thus, the AF-1 gene can be used in the preparation of tumor vaccines and other approaches for the gene therapy of cancer. As a diagnostic criteria, AF-1 can be measured in the serum or other body fluids such as urine to determine the level of cell deterioration. IFN-gamma is one of the few cytokines active on virtually all cells in all tissues of the body; and the IFN-gαmmα receptor and AF-1 is present on virtually all cells as well. General breakdown of cells as occurs in malignancy and other diseases can be measured by studying AF-1 in serum, urine, and other fluids. Gene therapy of immune cells as well as the tumor cells will provide a two-pronged approach to tumor therapy of other diseases. It is noted that little or no MHC class I antigen expression is often a characteristic of tumor cells that escape immune surveillance. The introduction of AF-1 into the immune and tumor cells can be used to reestablish normal function and elimination of malignant cells. Antibodies to AF-1 Polyclonal and monoclonal antibodies to AF-1 will provide diagnostic tests to measure AF-1 in fluids and on cells. As noted above, this can be used to establish immunoassays (ELISA, RIA, fluorometric, chemoluminescent, etc.) to measure AF-1. These antibodies may be able to be used directly to inhibit excess IFN-gαmmα, IFN-gamma receptor and/or AF-1 function in diseases where activation of the IFN-gamma system is causative in the disease process. Diseases in which a hyperactive IFN-gamma system is likely to include Type I diabetes, multiple sclerosis, lupus erythematosus, adjuvant arthritis, Shwartzman reaction, delayed type hypersensitivity and allotransplant rejection [see Garotta, G., Ozmen, L., and Fountoulakis, M. (1989) "Development of Interferon-gαmmα Antagonists as an Example of Biotechnology Application to Approach New Immunomodulators," Pharmacological Research 21 , Supplement 2, 5-17]. Soluble Forms of AF-1 Because antibodies can be used directly to block function of proteins, soluble forms of receptors and receptor components can be used to block receptor function and down regulate overactivity as in the diseases noted above. Soluble forms of receptors can be produced in prokaryotic or eukaryotic expression vectors, purified and used to down regulate over expression. If external contact with receptors is all that is required to generate a stimulation, then the soluble form of AF-1 can stimulate IFN-gamma function and serve as an activator directly without the necessity to enter the plasma membrane of the cell. Since the extracellular domain of the IFN-gamma receptor interacts with the extracellular domain of AF-1, the soluble forms may be able to carry out this activation directly. The soluble forms of AF-1 can be used to generate antibodies as could the intact complete molecules. Generating a Complete Functional Cell-free Receptor Soluble forms of the IFN-gamma receptor and other receptors have been shown to bind ligand with high affinity [see Fountoulakis, M. ,Juranville, J.- F.,Stuber, D., Weibel, E. K., and Garotta, G.( 1990) "Purification and Biochemical Characterization of a Soluble Human Interferon gamma Receptor Expressed in Escherichia coli," J. Biol. Chem. 265, 13268-13275; Fountoulakis, M., Juranville, J.-F., Maris, A., Ozmen, L., and Garotta, G. (1990) "One Interferon gamma Receptor Binds One Interferon gamma Dimer," J. Biol Chem. 265, 19758-19767]. However, it appears that the complete receptor requires AF-1 as well. Thus, an analysis of the structure activity relationships between receptor and ligand IFN- gamma will require AF-1 for refinement of the structural interfaces involved. This will lead to the development of small molecules that can activate the receptor and substitute for the large IFN-gαmmα protein ligand. Ideally, small molecules will be defined by molecular computer analysis of structure as well as crystallography to fill the interface and stimulate the receptors specifically. Furthermore, such molecules can be given orally in contract to parental injection that is now necessary of IFN-gamma. Small molecules can be designed to be agonists or antagonists of IFN-gαmmα function and thus can be used to mimic IFN-gamma or antagonize its actions for the syndromes noted above. Plasmids Manipulating YAC Clones and Their Uses The variety of plasmids we have developed to introduce markers into YACs, to delete regions of YACs and to fragment YACs provides new ways to manipulate YACs, introduce genes and locate genes within YACs. The plasmids have been designed for use with YAC containing human DNA insertions. Similar plasmids can be designed for manipulation of YACs from other animal species on the basis of the same principles. Transgenic Mice Containing AF-1 and the IFN-gamma Receptor Currently there is no way to test species specific human cytokines for true function in animal models. Efficacy and side-effects cannot be properly evaluated except in tedious and potentially hazardous human trials. The use of transgenic animals containing transgenes comprising the Hu-IFN-gαmmα receptor and AF-1 will provide the first step to generate mice that respond to human interferon just as we have shown than foreign cells expressing these components now respond to Hu-IFN-g mmα. Because the other accessory factors are defined fully and isolated, the entire Hu-IFN-g mmα system can be established in mice. Side effect and all actions of Hu-IFN-g mmα will be able to be assessed in animal models that will increase the safety of the ultimate testing in humans of IFN- gamma. Furthermore, the principles developed permit a basis for establishing procedure to test other species-specific cytokines in animals. Helper Function of Accessory Factors The accessory factors serve to assist and enhance functions through the IFN-gαmmα receptor. AF-1 has specifically defined functions to overcome species specificity in conjunction with the corresponding receptor. Just like other helper proteins, such as B7, AF-1 and the other accessory factors support and enhance IFN-gamma receptor function. The IFN-gamma system has provided many insights into this complex system, but it will be paralleled by the IFN-c. system as well. Thus, the IFN-α receptor complex will have multiple components that serve accessory functions for the IFN-α receptor. The present invention is further illustrated by the following examples which are not intended to limit the effective scope of the claims. All parts and percentages in the examples and throughout the specification and claims are by weight of the final composition unless otherwise specified. EXPERIMENTAL PROCEDURES YAC Clone Encoding an Accessory Factor Cells and Media The 16-9 cell line is a Chinese hamster ovary (CHO-K1) hybrid containing a translocation of the long arm of human Chromosome 6 and a transfected human HLA-B7 gene (14,15; A. Rashidbaigi, unpublished results). The original parental line, 836-3 A (11,14, 15), was subcloned to obtain cells which expressed the human IFN-gamma receptor as demonstrated by binding and cross- linking to 32 P-labeled Hu-IFN-gαmmα. These cells were then transfected with plasmid pJY105Rl.l, a genomic clone encoding the human HLA-B7 antigen, obtained from S. Weissman (35,36). The cells were treated with 15,000 units/mL of Hu-IFN-aA/D(Bgl), a human interferon active on hamster cells (37), for 2 days at 37°C. Then cells expressing the HLA-B7 antigen were isolated by treatment of cells with anti-HLA monoclonal antibody W6/32 (38) followed by incubation in a plate pre-coated with goat anti-mouse IgG (F c -specific). Unbound cells were removed by washing and the bound cells were pooled and then subcloned as described (15,25). Subclones were analyzed for the presence of the long arm of human Chromosome 6 by binding and cross-linking with p P]Hu-IFN-gαmmα and for HLA-B7 by a surface radioimmunoassay with the W6/32 monoclonal antibody (14,15,25). Clone 16 was positive for human Chromosome 6 and HLA-B7 expression. Clone 16 was then subcloned and retested. Subclone 16-9 was shown to contain human Chromosome 6 and demonstrated the highest level of HLA-B7 induction in response to Hu-IFN-αA/D. These cells were unresponsive to treatment with Hu-IFN-gαmmα. The 153B7-8 cells are CHO-K1 cells carrying human Chromosome 21q and the human HLA B7 gene (14,15). The 3xlS cells are CHO- Kl cells that contain a small 1-3 kb region of human Chromosome 21 (22). These cells are maintained in F12 (Ham) medium (Sigma) containing 10% heat-inactivated bovine serum (Sigma), and 50 μg/ml gentamicin sulfate (complete F12 medium). Ten YAC clones were obtained through the "Chromosome 21 Joint YAC Screening Effort" (directed by Dr. David Patterson, Eleanor Roosevelt Institute, Denver, CO, USA). YAC clones were maintained and grown in AHC media. AHC medium contains 1.7 g of yeast nitrogen base without amino acids, 5 g of (NH4)2SO4, 10 g of casein acid hydrolysate, 20 μg of adenine hemi-sulfate, and 20 g of glucose per liter. The addresses of the YAC clones used in this study are 525 B119G7, 525 A165D7, 525 C4C10, 525 C14B2, H8(517) A222A12, GART D142H8, 518 A234B10, 518 B134B9, SOD D112A5, and IFNAR B49F1. All YACs were derived from Sacchammyces cerevisiae AB1380 (MATa, ura3, trpl, adel- , cαwl-100, lyslΛ and his5) (27). Construction of Integrating Plasmid A 5.4 kb Hind ll restriction fragment containing the Sacchammyces cerevisiae LYS2 gene (a gift from Dr. Jules O'Rear, UMDNJ-Robert Wood Johnson Medical School) was inserted into the Hindlll site of pMClneo polyA (Stratagene, CA) to yield the plasmid pMC-LYS2. A 0.3 kb Alu family repeat in plasmid pBP47 (33) (a gift from Dr. Roger H. Reeves, Johns Hopkins University School of Medicine, Baltimore, MD) was modified by inserting a Clal linker into the EcOrR site to form plasmid pBP47-CYvI. The modified Alu fragment was cut from plasmid pBP47-C7αI with BamHI, treated with mungbean nuclease, and ligated to the Sail site of pMC-LYS2 after filling-in with the Klenow fragment of DNA polymerase I, yielding plasmid pJS l , the integrating plasmid. Construction of YACs Containing the Antibiotic G418 Resistance Marker Transformation was carried out as described (39) with the following modifications. Fifty milliliters of each YAC clone were grown to an absorption of 1.0 unit/mL at 600 nm in AHC liquid medium, harvested, and suspended in 5 mL of 1 M sorbitol. Fifty microliters of 10 mg/mL Zymolyase 20T (ICN) were added to the cell suspension, which was then incubated at 37°C for 15-30 minutes to obtain greater than 95% conversion to spheroplasts. The spheroplasts were washed three times with 1 M sorbitol, collected by centrifugation (500 x g, 5 minutes) and resuspended in STC medium (1 M sorbitol, 10 mM Tris/Cl pH 7.5, 10 mM CaC_2) at a density of 5 x 10° spheroplasts/mL. Five micrograms of linearized plasmid pJSl and 10 μg of salmon testes DNA as a carrier were put into 100 μL of the spheroplast suspension in a polypropylene tube, and the mixture incubated for 5 minutes at room temperature. After addition of 4 mL of polyethylene glycol (PEG) solution (20% w/v PEG 8000, 10 mM CaC_2, 10 mM Tris/Cl, pH 7.5), the mixture was incubated for 10 minutes at room temperature, then centrifuged (500 x g, 5 minutes) to pellet the spheroplasts. The spheroplasts were resuspended in 300 mL of SOS medium (1 M sorbitol, 5 mM CaCl2 in YPD medium consisting of 10 g of yeast extract, 20 g of peptone, and 20 g of glucose per liter), then incubated for 20 minutes at 30°C with shaking. Finally, the spheroplasts were mixed with 6 mL of top agar and the mixture was overlaid on a synthetic dextrose minimal medium plate supplemented with adenine sulfate (10 μg/mL) and histidine (20 μg/mlr) to select Lys + transformants. Fusion Each of the YAC clones containing the neo τ gene was fused to 16-9 cells by a modification of a procedure already described (33). Thirty-five milliliters of each clone were grown in AHC media to stationary phase. After being washed twice with 20 mL of 1 M sorbitol, cells were resuspended in 5 mL of a solution which contained 1 M sorbitol, 100 mM sodium citrate (pH 5.8), 10 mM EDTA, and 30 mM β-mercaptoethanol. Eighty microliters of a Zymolyase 20T stock solution (10 mg/mL) were added, and the mixture was incubated at 37°C for 20 minutes until 95% of the cells were spheroplasts. The spheroplasts were pelleted and washed twice in 5 mL of a solution of 1 M sorbitol in 10 mM Tris/Cl (pH 7.5), resuspended in 5 mL of the same solution, and counted. While the spheroplasts were being washed, the 16-9 cells were harvested by trypsinization, resuspended in serum free F-12 (Ham) medium (Sigma), and counted. Aliquots containing 4 x 10' spheroplasts were placed in 15-mL tubes and centrifuged at 500 x g for 5 minutes. The supernatant was removed from the spheroplast pellet and 5 mL of the cell suspension containing 2 x 10" 16-9 cells was added carefully. The cells were then centrifuged at 500 x g for 5 minutes. The supernatant was removed and 50 μL of serum-free F12 medium was added to resuspend the pellets. Five hundred microliters of 45% PEG 1500 solution (Boehringer Mannheim Biochemicals) containing 5% DMSO, 10 μM β-mercaptoethanol, and 5 mM CaC_2 were added to the mixed cell suspension. The cells were mixed by tapping the tube briefly, incubated for 2 minutes at room temperature, and diluted with 5 mL of serum-free F12 medium. This cell suspension was left at room temperature for 20 minutes, then centrifuged at 600 x g for 6 minutes. The resulting pellet was resuspended in 5 mL of complete F12 medium and plated at 10" 16-9 cells per 100 mm plate. Thirty-six hours later, plates were washed with phosphate-buffered saline (PBS) to remove dead cells and yeast, then refed with complete F12 medium containing 450 μg/mL of G418 (Gibco). Cells were fed as necessary, and resistant colonies typically appeared after 10 to 14 days. The G418-resistant colonies were pooled and expanded for further analysis. Electrophoresis and Hybridization Procedures Agarose plugs were prepared as described (40). Yeast cells were grown to stationary phase at 30 °C in 50 L of AHC media, washed twice with 25 mL of 50 mM EDTA (pH 8.0) by centrifugation at 3500 x g for 5 minutes, followed by resuspension at a density of 1 x 10 1U cells/ml in 50 mM EDTA. An equal volume of 1 % (w/v) InCert agarose (FMC BioProducts) in 50 mM EDTA (pH 8.0) and 20 μL of 10 mg/mL Zymolyase 20T per milliliter of agarose solution were added to the cell suspension and the mixture was put into the plug mould. Spheroplasts were made by pushing plugs out of the mould into 1 mL of 0.5 M EDTA (pH 8.0) containing 7.5% β-mercaptoethanol per plug and incubating overnight at 37°C with gentle shaking. The solution was removed and the plugs were rinsed twice with 50 mM EDTA (pH 9.25). The spheroplasts were lysed by transferring to Solution ESP [0.5 M of EDTA (pH 9.25), 1 % Sarkosyl (IBI), and 1 mg/mL proteinase K (Sigma)] and incubating for 24 hr at 50°C. The agarose plugs were analyzed by pulsed-field gel electrophoresis (PFGE) (CHEF, OWL Scientific Plastics, Inc). The gels measured 13 cm x 13 cm, consisted of 120 mL of 1 % agarose in TBE (0.045 M Tris/borate, pH 8.0, 2.5 mM EDTA), and were run at 170 V for 24 hr at 15 °C with a pulse time of 70 seconds. The DNAs were blotted onto Nytran (Schleicher & Schuell) by standard Southern blot procedures (24,25). DNA probes were labeled with [α- 32 P]dCTP (NEN, 3000 Ci/mmol) with random hexadeoxynucleotides as primers (41). Yeast Colony Hybridization Colony hybridization of yeast cells was carried out as described (39). Briefly, the strains to be tested were streaked onto Nytran membrane filters on AHC plates. The plates were incubated for 2 days, and the filters were removed, air-dried and placed onto Whatman 3 MM paper sequentially saturated with the following solutions; Solution A (1 M sorbitol, 20 mM EDTA, 50 mM dithiothreitol) for 15 minutes, Solution B (1 M sorbitol, 20 2mM EDTA, 15 mg/mL Zymolyase 20T) for 2 hr at 37°C, Solution C (0.5 N NaOH) for 7 minutes, Solution D (0.5 M Tris-Cl, pH 7.5, 10 x SSC) twice for 4 minutes each, and Solution E (2 x SSC) for 2 minutes (1 x SSC consists of 0.15 M NaCl and 0.015 M sodium citrate, adjusted to pH 7.0 with NaOH). The filter was baked and hybridized with the probe by standard procedures (24,25). Cytofluorographic Analysis of Cells for Expression of the HLA-B7 Surface Antigen Cells were seeded in 24- well plates at a density of about 25,000 cells/well (0.5-1 mL/well) and were treated with 1 ,000 units/mL of the appropriate IFNs for about 72 hr by which time the cells were nearly confluent. These HLA assays were performed in the absence of antibiotic G418 as it was found the presence of this antibiotic inhibited MHC induction (T.M. Mariano, unpublished observation). Cells were trypsinized, transferred to 1.5 mL tubes, and washed with complete F12 medium. HLA-B7 antigen was detected by incubating the cells with 15 μL of culture supernatant containing a saturating concentration of mouse anti- HLA antibody, W6/32 (38), for 30 minutes at 4°C. Cells were washed with complete medium and resuspended in 15 μL of fluorescein isothiocyanate- conjugated (FITC- conjugated) sheep anti-mouse IgG diluted to 80 g/mL and incubated for 30 minutes at 4°C, after which they were washed with complete medium and resuspended in 200 μL of cold complete medium for the immediate analysis of live cells. If cells had to be fixed for future analysis, they were washed once with PBS, resuspended in 15 μL of 3% (w/v) paraformaldehyde in PBS, and incubated 1 to 16 hrs at 4°C. The fixed cells were washed with PBS and finally resuspended in 200 μL PBS. Samples were analyzed on a Coulter Epics Profile Cytofluorograph with a 15-milliwatt Argon laser tuned to 488 nm. Forward and 90° angle light scatter as well as integrated log green fluorescence signals (with a 488 nm long-pass laser blocking filter and 525 nm band-pass filter in place) were collected and analyzed. For each analysis, 10,000 events were accumulated and analyzed on the CytoLogic software as described (15,25). Antiviral Assay Transfected cells were assayed for resistance to encephalomyocarditis virus (EMCV) by a cytopathic effect inhibition assay (42). A/u-targeting YAC Deletion Plasmids Materials and Methods Strains GART YAC D142H8 was obtained from Dr. David Patterson (Eleanor Roosevelt Institute, Denver, CO. USA, Chromosome 21 Joint YAC Screening Effort), and propagated in AHC medium. AHC medium contains 1.7 g of yeast nitrogen base without amino acids and ammonium sulfate, 5.0 g of (NH4)2SO4, 10 g of casein acid hydrolysate, 20 μg of adenine hemi-sulfate and 20 g of glucose per liter. The human/hamster somatic cell hybrid line, 16-9, which contains human chromosome 6q and the human HLA B7 gene (14,57), was used for fusion with the YAC clones. The YAC clone is derived from S. cerevisiae AB1380 (Λ___7a, ade2- 1, canl-100, trpl, ura3, his5, [psi+]). Construction of Integrating Plasmid A 5.4 kb Hindlll restriction fragment containing the S. cerevisiae LYS2 gene (a gift from Dr. Jules O'Rear, UMDNJ-Robert Wood Johnson Medical School) was inserted into the Hindlll site of pMClneo polyA (Stratagene, CA) to yield the plasmid pMC-LYS2. A 0.3 kb BLUR 13 Alu family repeat in plasmid pBP47 (33) (a gift from Dr. R.H. Reeves, Johns Hopkins University School of Medicine, Baltimore, MD) was modified by inserting a Clάl linker into the EcøRI site to form plasmid pBP47-C/αI. The modified Alu fragment was cut from plasmid pBP47-C/αI with BamHI, treated with mungbean nuclease, and ligated to the Sail site of pMC-LYS2 after filling- in with the Klenow fragment of DNA polymerase I, yielding plasmid pJSl, the integration/deletion plasmid. Transformation of YAC Clones with the Integration/Deletion Plasmid Transformation was carried out as described (39) with the following modifications. Fifty milliliters of each YAC clone were grown to an absoφtion of 1.0 unit/ml at 600 nm in AHC liquid media, harvested, and suspended in 5 mL of 1 M sorbitol. Fifty microliters of 10 mg/ml Zymolyase 20T (ICN) were added to the cell suspension followed by a 15-30 minutes incubation at 37°C to get more than 95% conversion to spheroplasts. The spheroplasts were washed three times with 1 M sorbitol, collected by centrifugation (500 x g, 5 minutes) and resuspended in STC medium (1 M sorbitol, 10 mM Tris/Cl pH 7.5, 10 mM CaC_2) at a density of 5 x 10° spheroplasts/mL. Five micrograms of C/./I-linearized plasmid pJSl and 10 μg of salmon testes DNA as a carrier were put into 100 μL of the spheroplast suspension in a polypropylene tube, after which the mixture was incubated for 5 minutes at room temperature. Four milliliters of polyethylene glycol (PEG) solution (20% w/v PEG 8000, 10 mM CaCl 2 , 10 mM Tris/Cl, pH 7.5) were added and the mixture was incubated for 10 minutes at room temperature, centrifuged (500 x g, 5 minutes), resuspended in 300 mL of SOS medium (1 M sorbitol, 5 mM CaCl2 in YPD medium consisting of 10 g of yeast extract, 20 g of peptone, and 20 g of glucose per liter), then incubated for 20 minutes at 30°C with shaking. Finally, the spheroplasts were mixed with 6 mL of top agar and the mixture was overlaid on a plate of synthetic dextrose minimal medium supplemented with 10 μg/ml adenine sulfate and 20 μg/ml histidine to select Lys + transformants. Fusion of Mammalian Cells with Yeast Spheroplasts Each of the YAC clones containing the neo τ gene was fused to 16-9 cells by a modification of a procedure already described (54,33,48). Twenty milliliters of each clone were grown in AHC media to stationary phase. Four milliliters of the culture was diluted four times into YPD medium and grown at 30°C for one hour. After being washed twice with 10 mL of 1 M sorbitol, cells were resuspended in 3 mL of a solution which contains 1 M sorbitol, 100 mM sodium citrate (pH 5.8), 10 mM EDTA, and 30 mM β-mercaptoethanol. Forty microliters of a 10 mg/mL Zymolyase 20T stock solution was added, and the mixture was incubated at 37°C for 20 minutes until 95% of the cells were spheroplasts. The spheroplasts were pelleted and washed twice in 5 mL of a solution of 1 M sorbitol in 10 mM Tris/Cl (pH 7.5), resuspended in 3 mL of the same solution, and counted. While the spheroplasts were being washed, the 16-9 cells were harvested by trypsinization, resuspended in serum free F-12 (Ham) medium (Sigma), and counted. Aliquots containing 6 x 10 spheroplasts were placed in 15-mL tubes and centrifuged at 500 x g for 5 minutes. The supernatant was removed from the protoplast pellet and 5 ml of the cell suspension containing 3 x 10" 16-9 cells was added carefully. The cells were then centrifuged at 500 x g for 5 minutes. The supernatant was removed and 50 μL of serum-free F12 medium was added to resuspend the pellets. Five hundred microliters of 45% (w/v) PEG 1500 solution (Boehringer Mannheim Biochemicals) containing 5% (v/v) DMSO, 10 μM β-mercaptoethanol, and 5 mM CaCl2 were added to the mixed cell suspension. The cells were mixed by tapping the tube briefly, incubated for 2 minutes at room temperature, then diluted with 5 mL of serum-free F12 medium. This cell suspension was left at room temperature for 20 minutes, then centrifuged at 600 x g for 6 minutes. The resulting pellet was resuspended in 5 mL of complete F12 medium and plated at 1.5 x 10" eukaryotic cells per 150 mm plate. Thirty- six hours later, plates were washed with phosphate- buffered saline (PBS) to remove dead cells and yeast, then refed with complete F12 medium containing 450 μg/mL of antibiotic G418 (Gibco). Cells were refed as necessary, and resistant colonies typically appeared after 14 to 18 days. Electrophoresis and Hybridization Procedures Agarose plugs were prepared as described (40). Yeast cells were grown to stationary phase at 30 °C in 50 mL of AHC media, washed twice with 25 mL of 50 mM EDTA (pH 8.0) by centrifugation at 3500 x g for 5 minutes, followed by resuspension at a density of 1 x 10 10 cells/ml in 50 mM EDTA. An equal volume of 1 % (w/v) Incert agarose (FMC BioProducts) in 50 mM EDTA (pH 8.0) and 20 μL of 10 mg/mL Zymolyase 20T per milliliter of agarose solution was added to the cell suspension and eighty microliters of the mixture was put into the plug mould. Spheroplasts were made by pushing plugs out of the mould into 1 mL of 0.5 M EDTA (pH 8.0) containing 7.5% β-mercaptoethanol per plug and incubating overnight at 37°C with gentle shaking. The solution was removed and the plugs were rinsed twice with 50 mM EDTA (pH 9.25). The spheroplasts were lysed by transferring to Solution ESP [0.5 M EDTA (pH 9.25), 1 % Sarkosyl (IBI), and 1 mg/mL proteinase K (Sigma)] and incubating for 24 hr at 50°C. For restriction enzyme digestion one quarter of a plug was treated twice with one milliliter of 1 mM phenylmethylsulfonyl fluoride (PMSF) in TE [10 mM Tris/Cl (pH 7.5), 0.1 mM EDTA] at room temperature to inactivate proteinase K. Then the plug was washed three times with the same TE buffer for 2 hour each and put into a 1.5 milliliter microtube along with reaction buffer and 5 units of Nøtl restriction endonuclease (New England Biolabs) in a volume of 100 μl. The mixture was incubated at 37 °C overnight. The agarose plugs were analyzed by pulsed field gel electrophoresis (PFGE) (CHEF, OWL Scientific Plastics, Inc). The gels measured 13 cm x 13 cm, consisted of 120 mL of 1 % agarose in TBE (0.045 M Tris/borate, pH 8.0, 2.5 mM EDTA), and were run at 170 V for 24 hr at 15°C with a pulse time of 35 seconds. The DNAs were blotted onto Nytran (Schleicher & Schuell) with standard Southern blot procedures. DNA probes were labeled with [α- 32 P]dCTP (NEN, 3000 Ci/mmol) with random hexadeoxynucleotides as primers (41). Rescue of DNA Fragments Containing Alu Targeted Sites Chromosomal DNA was prepared from the transformed YAC clones as described (39). The DNA was digested with restriction endonuclease Xhol, separated on a 0.8% agarose gel, blotted onto a nylon membrane, and probed with a radiolabeled neo gene fragment in order to identify the size of Xhol fragment containing the neo gene. The appropriate DNA band was excised from the agarose gel and purified with Geneclean (BiolOl , La Jolla, CA). The DNA fragments were ligated to Xtol-digested, and alkaline phosphatase-treated pBluescript KS+/- (Stratagene). The resulting plasmids were transformed into E. coli INV/ which were then plated at a density of about 300 colonies per 150 mm Petri dish containing LB medium and 50 μg/ml ampicillin. The colonies were blotted onto nylon membranes and probed with the radiolabeled neo gene fragment to identify clones having one end of the integration site. Construction of Cosmid Library from YAC Clones A high molecular weight chromosomal DNA was made from YAC D142H8 as described (39). Ten micrograms of the DNA were incubated with 0.5 unit of restriction endonuclease Mbό (BRL) in a 100 μl volume. After 1, 5, 10, 20, and 30 minutes incubation, 20 μl of the mixture was taken and run on field inversion gel electrophoresis (FIGE) with standard program No. 3 (PPI-200, M. J. Research Inc.) which is suitable to resolve DNA up to 200 kb. A ten minute digestion was chosen since it yielded a 50 to 100 kb size distribution. Then 10 μg of the DNA was digested with Mbol restriction endonuclease as described above for 10 minutes, extracted with phenol and phenol/chloroform (1: 1), treated with alkaline phosphatase, and precipitated with ethanol. Five μg of the cosmid vector SuperCosl (Stratagene) was cut with endonuclease Xbal, treated with alkaline phosphatase, and cut again with endonuclease BamHI to get two fragments (1.1 kb and 6.5 kb). Then 2.5 μg of digested chromosomal DNA and 1 μg of digested SuperCosl were ligated in a 20 μl volume. After ligation, 2 μl of the DNA mixture was packaged with extract (Gigapack II plus, Stratagene) according to the manufacturer's procedure. The infected E. coli AGl cells were plated onto LB plates containing 50 μg/ml of ampicillin at a density of about 300 colonies per 150 mm Petri dish for screening. Yeast Artificial Chromosome Fragmentation Vectors Materials and Methods Yeast Strains Yeast host strain AB1380 containing a 540 kb YAC ('B8') and was grown in AHC medium (1.7 g yeast nitrogen base without amino acids, 5.0 g of (NH4)2SO4, 10 g of casein hydrolysate, 20 μg adenine hemisulfate and 2% (w/v) glucose per liter). This host strain was transformed to a Tφ + , Ura + phenotype by introduction of the GART YAC. The compound 5-fluoro-orotic acid (5-FOA, obtained from PCR, Inc., Gainesille, FL) was used to obtain Ura " clones according to the method of McCusker and Davis (63). The Ura strain containing the B8 YAC was plated on 2% agar plates containing 0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.1 % proline. 2% glucose, 10 μg/ml uracil, 50 μg/ml 5-FOA, 10 μg/ml adenine sulfate, 50 μg/ml lysine and 20 μg/ml histidine to obtain URA3 " cells. Potential ura3 " mutants were picked, streaked for single colonies, and characterized with regard to growth without uracil. A clone containing the B8.ura.2 YAC was selected for further use. Vector Construction A plasmid containing the human repetitive Alu sequence Blur-13 (pBP47, obtained from Dr. Roger H. Reeves, Johns Hopkins Univ.) was modified by removal of the EcøRI site. After digestion with restriction endonuclease EcøRI, the ends were filled in with the Klenow fragment of DNA polymerase I. An 8 bp Clal linker was then inserted at this location, adding a total of 12 bp to the Alu sequence. This modified Blur- 13 fragment of about 300 bp was released with restriction endonuclease BαmHI, made blunt-ended with the Klenow fragment of E. coli DNA polymerase I and isolated with Geneclean Plus (Bio 101, La Jolla, CA). This fragment was ligated into the S al site of pBluescript KS+ and clones containing the Blur-13 fragment in both orientations were selected. The orientation of the Blur-13 fragment was determined by using an asymmetric Bg\I site located at position 109 in the Blur-13 fragment. Following this, the iJαmHI site was removed from the polylinker in these clones by digesting with the restriction endonuclease 2-αmHI and filling in the 5'- overhang with the Klenow fragment of E. coli DNA polymerase I. The Blur- 13 fragments were released from these modified Bluescript vectors with EcόSl and Nøtl restriction endonucleases. The EcøRI-Nøtl fragments, containing Blur fragments in both orientations, were then cloned into the plasmid pYAC4DRI+Supll, SV2ΝEO (a gift from Dr. Jules O'Rear, Robert Wood Johnson Medical School, UMDNJ) to form pSEl and pSE2, differing only in the orientation of the Blur sequence (Figure 10). Yeast Transformation The yeast strain containing the YAC B8.ura.2 was transformed to Ura + with the fragmentation vectors pSEl and pSE2. First, DNA was prepared from each of these vectors by digestion with EcøRI and BamHI restriction enzymes. The DNA fragments were then dephosphorylated with calf intestinal alkaline phosphatase. The protein was denatured with plienol/chloroform/isoamyl alcohol (25:24: 1) extraction three times, and the DNA was precipitated with ethanol. Twenty-five μg of DNA (a mixture of 12.5 μg of each plasmid) was used to transform 5 x 10 7 yeast cells containing YAC B8.ura.2 as reported (39). Ura + transformants were selected on uracil-deficient plates and 27 clones were chosen for analysis. Transformation of Mammalian Cell- Chinese hamster ovary (CHO) cells were transformed by neo + yeast according to Pavan et al. (33). Briefly, approximately 2x10' CHO cells were fused to 5 ml of a confluent yeast culture as described (33). After 24-36 hr in F-12 medium plus 10% fetal bovine serum, the cells were washed twice with PBS. Transformants were selected by 450 μg/ml of G418. Localization of the Human Interferon-gαmmα Receptor Accessory Factor Gene Cells The hamster/human somatic cell hybrid 16-9 which contains human Chromosome 6q was maintained in F-12 medium containing 10% fetal bovine serum. These cells had been transfected with a genomic clone for the class I HLA- B7 antigen (98,105). The cell lines which were transformed by fragmented YACs were maintained in the same medium plus 400 μg/ml G418. The 153B7-8 cell line, defined previously (98) is a Chinese hamster ovary (CHO-K1) cell line which contains human Chromosome 21q, and the HLA-B7 antigen gene; 153B7-8[HHH] refers to the same cell line which has been transfected with the Hu-IFN-gαmmα R cDNA. Both of these cell lines were maintained as described earlier (101,107). The 3xlS cell line is a CHO-Kl cell line which contains 1-3 mb of human Chromosome 21 (104). Reagents, restriction endonucleases and other enzymes. Human IFN-gamma, with a specific activity of 1.3 x 10' units/mg, was isolated from Escherichia coli as described (108). Human IFN-α/A/D(Bgl) was prepared as reported (109,110) and had a specific activity of 6.0 x 10 7 units/mg. Human IFN-gamma was isolated as described (111 ,112) and had a specific activity of 3.5 x 10 units/mg. Restriction endonucleases were obtained from New England Biolabs and Boehringer Mannheim. Construction of fragmented YACs The parental GART YAC (GART D142H8) was obtained through the "chromosome 21 joint YAC screening effort" (directed by Dr. David Patterson, Eleanor Roosevelt Institute, Denver, CO). The plasmids pSEl and pSE2 were constructed in such a way that BamHI and EcøRl digestion releases an 8.4 kb fragment containing an Alu sequence, an SV2neo gene, a URA3 gene and a yeast telomere, in that order (106,113). The plasmids pSEl and pSE2 (12.5 μg of each) were so digested and the 8.4 kb fragment was isolated and used to transform the yeast strain GART D142H8.ura.2 which contains the parental GART YAC and had been converted to a Ura " phenotype by 5-fluoroorotic acid selection (106). Transformations were performed as described (113) and Ura transformants were obtained on uracil-deficient plates. Fusion of fragmented YACs to mammalian cells Approximately 2 x 10' 16-9 cells were fused to 10 ml of a 5% confluent yeast culture with PEG (113). After 24-36 hours in F-12 medium plus 10% fetal bovine serum and 50 μg/ml gentamycin, the cells were washed twice with phosphate-buffered saline (PBS) and the transformants were selected in the same medium containing 450 μg/ml antibiotic G418. MHC analysis Induction of MHC class I antigens by IFN was assayed as reported (101,110). For each sample, 10,000 cells were analyzed with a Coulter Epics Profile cytofluorograph and Cytologic software. Assay of the Transcription Factors ISGF3-gαmmα and GAF For factor ISGF3-gαmmα, cells transformed by fragmented YACs were incubated with or without 100 units/ml IFN-gαmrnα for 18 hours. The cells were harvested by trypsin-EDTA treatment, washed once in F-12 plus 10% fetal bovine serum and once in PBS. Lysates were prepared from the cell pellets and gel shift assays were conducted as described (114). GAF was assayed as described (115) after cells had been incubated with or without 100 units/ml IFN-gamma for 30 minutes. Antiviral assays The cell lines 16-9, J18/16-9, J29/16-9 and 153B7-8[HHH] were assayed for resistance to encephalomyocarditis virus (EMCV) as detailed previously (110,116). Forty thousand cells were plated per well and EMCV was present at 4,000 pfu/well. cDNA Clone Encoding an Accessory Factor Reagents, Restriction Endonucleases and Other Enzymes All restriction endonucleases were purchased from New England Biolabs; DNase I was from Worthington Biochemicals; Sequenase was from United States Biochemical Coφoration; T4 DNA ligase was from Boehringer Mannheim. [α-^PJdCTP and [gαmmα- 32 P]ATP were from New England Nuclear. Acrylamide, formamide and all other reagents were analytical grade and purchased from United States Biochemical Coφ. Cells and Media The 16-9 hamster/human somatic cell hybrid line is a Chinese hamster ovary (CHO-Kl) hybrid containing a translocation of the long arm of human Chromosome 6 and a transfected human HLA-B7 gene (14,15,65). These cells are unresponsive to treatment with Hu-ΪFN-gamma. The 3xlS cells are CHO- Kl cells that contain a small 1-3 mb region of human Chromosome 21 (65,22,46). The 16-9 cells were maintained in F12 (Ham) medium (Gibco) containing 10% heat-inactivated fetal bovine serum (Sigma), and 50 μg/ml gentamicin sulfate (complete F12 medium). The 3xlS cells were maintained in F12D (Ham) medium (Gibco) containing 10% dialyzed heat-inactivated fetal bovine serum (Sigma), and 50 μg/ml gentamicin sulfate (complete F12D medium). YACs were obtained, maintained and grown in AHC media as reported (65). Isolation of RNA and DNA Total polyadenylated RNA was isolated from Raji, HL60 and 3xlS cells as described (66). DNA was prepared by minor modification of previously reported procedures (24,67-69). High molecular weight DNA was prepared from Daudi cells and human placenta by the procedure of Blin and Stafford (69) Construction of Cosmid Library from YAC Clones and Genomic Walking Genomic cosmid libraries from 3xlS cells and human cells were prepared as described (46). A high molecular weight chromosomal DNA was made from YAC D142H8 (65) or J18 (70,71) as described (39). Ten micrograms of the DNA was incubated with 0.5 unit of restriction endonuclease Mbøl (BRL) in a 100 μl volume. After 1, 5, 10, 20, and 30 minutes incubation, 20 μl of the mixture was taken and run on field inversion gel electrophoresis (FIGE) with standard program No. 3 (PPI-200, M. J. Research Inc.) which is suitable to resolve DNA up to 200 kb. A ten minute digestion was chosen since it yielded a 50 to 100 kb size distribution. Then 10 μg of the DNA was digested with Mbόl restriction endonuclease as described above for 10 minutes, extracted with phenol and phenol/chloroform (1:1), treated with alkaline phosphatase, and precipitated with ethanol. Five μg of the cosmid vector SuperCosl (Stratagene) was cut with endonuclease Xbal, treated with alkaline phosphatase, and cut again with endonuclease BαmHI to get two fragments (1.1 kb and 6.5 kb). Then 2.5 μg of digested chromosomal DNA and 1 μg of digested SuperCosl were ligated in a 20 μl volume; two μl of the ligated DNA mixture was packaged with extract (Gigapack II plus, Stratagene) according to the manufacturer's procedure. The infected E. coli AGl cells were plated onto LB plates containing 50 μg/ml of ampicillin to a density of about 300 colonies per 150 mm Petri dish for screening. About 150 cosmid clones which were positive to human Cot 1 DNA (BRL) were selected. Then Cot 1 positive cosmids were screened with the radiolabeled TRPl and URA3 gene fragments to identify the cosmids GC3-59 and JC8 corresponding to YAC centric and acentric arms, respectively. A 1.5 kb fragment from the T3 RNA polymerase end of the cosmid GC3-59 free of repetitive human DNA was used to identify the overlapping cosmids GC8-3 and GC8-10. A 700 bp fragment free of repetitive human DNA from the T7 end of the cosmid GC8-10 was used to identify the cosmid JC35 (Figure 12). Preparation and Screening of cDNA Libraries; Sequencing of DNA A cDNA library from 3x lS cells was constructed in phage lambda pCEV27 (72) by the automatic directional cloning method (73). The library consisted of 1.5 x 10 7 independent clones and nearly all phages contained cDNA inserts. A part of the library was amplified and used for screening. Previously prepared libraries from mRNA from M426 human cells in lambda pCEV15 (73) and from mRNA from human Daudi lymphoblastoid cells (plasmid pCDNAl custom library prepared by Invitrogen; a gift from J.A. Langer) were used as well. Screening of cDNA and genomic libraries with DNA probes was performed as described previously (24). The cDNA clones were sequenced by a modification of the dideoxy chain termination method (74,75). SP6, T7 and internal primers generated from the sequence were used to prime the sequencing reactions with double-stranded templates. Hybridization Procedures DNA used as probes in the hybridization reactions represented the entire insert of the cDNA clones unless otherwise noted and was labeled by the random oligonucleotide primer labeling method according to standard techniques (68,41). All hybridization reactions were carried out in 50% formamide, 5 x SSC, 5 x Denhardt's reagent, 0.1 % SDS, 100 μg/ml yeast tRNA and 0.1 μg/ml of poly(A) (Collaborative Research, Inc.) overnight (about 16 hours) at 42°C. The hybridization blots were washed stringently with 0.1 x SSC, 0.1 % SDS at 65 °C. In order to determine the size of the accessory factor mRNA, 1 μg of polyadenylated RNA from various cells was fractionated on a 1.2% agarose formaldehyde gel and blotted onto a nylon membrane (Nytran, Schleicher and Schuell). For Southern blots, DNA (10 μg) was digested with EcøRI and Hindlll restriction endonucleases. The restriction digests were electrophoresed on a 20 cm 0.8% agarose gel and blotted onto the nylon membrane. The Northern and Southern blots were hybridized with 32 P-labeled DNA under conditions described above. The blots were washed stringently, as desribed above, then autoradiographed with Kodak XAR-5 film and a Cronex Lightning Plus intensifying screen (DuPont) at -70 °C for 48 hours. To suppress signals from repetitive human sequences, 25 ng of the genomic DNA fragment was labeled by random priming (41) to high specific activity (10 cpm/μg) followed by denatu ration of the probe in a large excess of sheared human DNA (1 mg/ml) (human Cotl DNA, GibeoBRL) , and pre- annealing at 68°C in 6 X SSC for 10 minutes to Cot 10 (76). Computer Analysis of Sequence Data The Wisconsin GCG software package (77) was used to assemble a composite sequence from the sequences of the various fragments and for other computer analyses of the data. Optimal alignments between the sequences were made by the algorithm of Needleman and Wunsch (78). The hydrophobicity plot of the deduced amino acid sequence was done according to the method of Kyte and Doolittle (79). Transfection of 16-9 Cells Transfections were performed as previously described (17,25,68). After transfection, the cells were incubated for 20 hours at 35 °C in a 3% CO2 atmosphere, after which the medium was removed, the cells were washed twice with phosphate-buffered saline, and incubated in fresh medium in 5% CO2 at 37 °C for 20 hours. The cells were then trypsinized, split at a ratio of 1: 10, and incubated in medium under the same conditions for 20 hours before selection by resistance to antibiotic G418 (450 μg/ml). Cytofluorographic Analysis of Cells for Expression of the HLA-B7 Surface Antigen Cytofluorographic analysis of cells for expression of the HLA-B7 surface antigen was performed as described in detail previously (17,20,65,25). Clones or pools of cells were analyzed as noted. Samples were analyzed on a Coulter Epics Profile Cytofluorograph with a 15-milliwatt Argon laser tuned to 488 nm. Forward and 90° angle light scatter as well as integrated log green fluorescence signals (with a 488 nm long-pass laser blocking filter and 525 nm band-pass filter in place) were collected and analyzed. For each analysis, 10,000 events were accumulated and analyzed on the CytoLogic software as described (80,25). Hu-IFN-αA/D, a human interferon active on hamster cells (80), was used as a control to demonstrate the integrity of the HLA-B7 gene in the hamster cells. Antiviral Assay Transfected cells were assayed for resistance to encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) by a cytopathic effect inhibition assay (42). Table 1 Functional Assay of YAC Clones Locus Probe Address Size of AC.neo 1* No of MHC Name of YAC YAC(kb) G418 Response Resistant Colonies D21S65 525 B119G7 1200 525 B119G7.neo.1 2 525 B119G7.neo.18 0 NA D165D7 210 525 A165D7.neo.12 0 NA 525 A165D7.neo.16 0 NA C4C10 220 525 C4C10.neo.3 0 NA 525 C4C10.neo.20 0 NA C14B2 450 525 C14B2.neo.2 0 NA 525 C14B2.neo.19 0 NA D21S17 H8 A222A12 220 H8(517)A222A12.neo.6 0 NA (517) H8(517)A222A12.neo.13 3 - D21PFGS GART D142H8 540 GARTD142H8.neo.3 4 + GARTD142H8.neo.16 1 GARTD142H8.neo.18 many + D21S55 518 A234B10 210 518A234B10.neo.2 0 NA 518A234B10.neo.15 0 NA B134B9 225 518B134B9.neo.8 0 NA D21S0D1 SOD D112A5 180 0 NA 120 0 NA D21IFNAR IFNAR B49F1 150 IFNARB49F1.neo.32 5 IFNARB49F1.neo.35 0 NA The "MHC Response" of the last column represents Human class I HLA antigen induction upon treatment with Hu-IFN-gαmmα: - represents no response; + represents a positive response; NA, not applicable. Transfected cells were tested as pools of G418-resistant colonies. The SOD D112A5 YAC was not tested for HLA induction since a weø r -positive YAC was not obtained from the transformation. The dish of transformants with GARTD142H8.neo.18 (YAC-JS2) contained many small colonies. Table 2 Antiviral Effects of Ku- N-gamma on Transfected CHO cells Cell Line DNA Transfected IFN ED50 (units/ml) 153B7 - 8 Hu-IFN-gαmmα R cDNA Hu-IFN-gαmmα 2 3xlS Hu-IFN-gαmmα R cDNA Hu-IFN-gαmmα 14 16 - 9 YAC-JS2 Hu-IFN-gαmmα > 10000 Transfected cells were assayed for resistance to EMCV by a cytopathic effect inhibition assay (42). The 153B7-8 cells are CHO-Kl cells containing a transfected HLA-B7 gene and human Chromosome 21q (15, 25). Irradiation- reduced somatic cell hybrid 3xlS cells containing a 1-3 mb fragment of human Chromosome 21q were described (22). The somatic cell hybrid 16-9 cells contain human HLA-B7 gene and human Chromosome 6q encoding the Hu-IFN-gαmmα receptor. The ED50 represents the concentration of IFN-gamma (units/ml) which provided 50% protection of cells against EMCV. All the cells were protected from EMCV infection by Hu-ΪFN-aA/D(Bgl) in control experiments. Each of these cell lines were derived from the same parental CHO-K 1 cells. Table 3 Distribution of the Size of Deletions. Size of Deletion (kb) No. of Deleted Clones (%) 0 - 20 38 (63.3%) 20 - 100 17 (28.3%) > 100 5 (8.3%) Table 4 Characteristics of fragmented YACs produced by URA3 transformation YAC Characteristics Percentage Fraction YACs < 540 kb78% 78% (21/27) YACs > 540 kb 11 % (3/27) URA clones with no apparent YAC 11 % (3/27) 80 kb < YACs < 150 kb 48% (13/27) 150 kb < YACs < 350 kb 7% (2/27) 350 kb < YACs < 540 kb 22% (6/27) YACs positive for GART gene 59% (16/27) YACs positive for GC8.10E6 89% (24/27) YACs were analyzed by pulsed-field gel electrophoresis at a field strength of 170 V. Their size was determined relative to Saccharomyces cerevisiae chromosomal DNA standards. YACs selected for further mapping include J29 (500 kb), J28 (475 kb), J16 (350 kb), and J18 (150 kb). The genotype of these YAC strains is neo + , TRPl, URA3. Table 5 Amino Acid Composition of Human Interferon gamma Accessory Factor-1 Complete Protein Mature Protein Residue Number Mole Number Mole Percent Percent Ala 25 7.4 19 6.1 Cys 6 1.8 6 1.9 Asp 16 4.7 15 4.8 Glu 16 4.7 16 5.2 Phe 18 5.3 17 5.5 Gly 14 4.1 13 4.2 His 6 1.8 6 1.9 He 20 5.9 20 6.5 Lys 13 3.9 13 4.2 Leu 37 11.0 29 9.4 Met 5 1.5 4 1.3 Asn 11 3.3 11 3.5 Pro 25 7.4 21 6.8 Gin 17 5.0 17 5.5 Arg 12 3.6 11 3.5 Ser 31 9.2 30 9.7 Thr 20 5.9 19 6.1 Val 24 7.1 23 7.4 Tip 9 2.7 8 2.6 Tyr 12 3.6 12 3.9 Total 337 100 310 100 Ala + Gly 39 11.6 32 10.3 Ser+Thr 51 15.1 49 15.8 Asp + Glu 32 9.5 31 10.0 Asp + Glu + Asn + Gin 60 17.8 59 19.0 His + Lys + Arg 31 9.2 30 9.7 Asp + Glu + His + Lys + Arg 63 18.7 61 19.7 Ile+Leu+Met+Val 86 25.5 76 24.5 Phe+Trp+Tyr 39 11.6 37 11.9 The amino acid composition of the coding sequence of human IFN-gαmmα AF-1 from positions 1-337 (complete pre-protein) and 28-337 (the estimated mature protein) are given. The molecular weight of the polypeptide including the signal peptide is 37,834; the mature protein, 35,034. The isoelectric point of the mature protein is calculated to be pH 5.42. The analysis presents the coding sequence of plasmid pSKl. Table 6 Homology of the Class 2 Receptor Family to Human AF-1 Receptor Amino Acids in Domain % Identity % Similarity Human CRF2-4 201 29.6 48.7 Human IFN-gαmmα R 228 21.0 42.9 Murine IFN-gamma R 227 25.8 46.4 Human IFN-gαmmα R N-term 197 25.0 50.0 C-term 212 24.9 49.2 Murine IFN-gamma R N-term 198 22.4 48.4 C-term 220 23.5 42.6 Bovine IFN-gamma R N-term 200 25.8 46.8 C-term 200 22.7 40.1 Percent homology to the extracellular domain of the human IFN-gamma AF-1 mature protein as determined by the alignment method of Gribskov and Burgess (93) as written for the GCG Sequence Analysis Software Package (Madison, WI). Table 7 Antiviral (EMCV) Activity of Interferons in Cell Lines Stably Fused with YACs Containing the Human Chromosome 21 Accessory Factor Gene IFN Concentration (units/ml) Providing Half-maximal Protection Interferon 16-9 Cells J 18/ 16-9 Cells J29/ 16-9 Cells 153B7-8 [HHH] Hu-IFN-c.A/D(Bgl) 528 264 378 N.D. Hu-IFN-β 700 172 112 N.D. Hu-IFN-gαmmα (> 3,250) (> 3,250) (> 13,000) 3.2 N.P. N.P. N.P. Interferons were tested over the following concentration ranges: For 16-9 cells (the untransfected host) and J- 18/ 16-9 cells, all interferons were tested from 0.06 to 250 ng/ml. For J29/16-9 cells, the interferons were tested from 0.24 to 1,000 ng/ml. Specific activity of the interferons used was: Hu-IFN-<_A/D(Bgl), 6.0 x 10 7 units/mg; Hu-IFN-β, 3.5 x 10 7 units/mg; Hu-IFN-gαmmα, 1.3 x 10 7 units/mg. N.P. designates no protection observed at maximal Hu-IFN-gαmmα concentration tested; N.D., not done. HHH indicates that the Hu-IFN-gαmmα receptor cDNA was transfected into the cell. Figure 1 shows a pulse field gel electrophoresis of various YACs and hybridization to total human DNA. Panel A: PFGE analysis of 9 YAC clones from the 3xlS region of human Chromosome 21q. Lane 1, S. cerevisiae AB1380; lane 2, 525 B119G7 (1200kb); lane 3, 525 A165D7 (210 kb); lane 4, 525 C4C10 (220 kb); lane 5, 525 C14B2 (450 kb), lane 6, H8(517) A222A12 (220 kb); lane 7, GART D142H8 (540 kb); lane 8, 518 A234B10 (210 kb); lane 9, 518 B134B9 (225 kb); lane 10, SOD D112A5 (180 kb, 120 kb). IFNAR B49F1 (150 kb) is not shown here. Panel B: Southern hybridization of the PFGE gel. The blot from Panel A was probed with total human DNA labeled by random priming. The size of each YAC is shown above in parentheses. The numbers to the right of the blot indicate the size of various fragments in kilobase pairs (kb). Figure 2 illustrates a Southern hybridization. A: The agarose plugs from neo τ Lys 2 transformants derived from each YAC clone were analyzed by PFGE and the blot was probed with the labelled neo τ gene to determine whether or not the neo τ gene was targeted to the YAC. Lane 1, GART D142H8; lane 2, GART D142H8.neo.16; lane 3, GART D142H8.neo.18; lane 4, IFNAR B49F1 ; lane 5, IFNAR B49Fl.neo.32; lane 6, IFNAR B49Fl.neo.35 YACs are shown here as example. The GART D142H8.neo.18 (lane 3) clone contains smaller YACs than the original one (GART D142H8), possibly resulting from deletions generated by homologous recombination between two Alu sequences on the original YAC during the integration process. B: Yeast chromosomal DNAs from the same YAC clones as in A were digested with 2-αmHI and Xhol to release the neo T gene cassette and the blot was probed with the labelled neo τ gene. Each targeted YAC clone shows a 1.1 kb fragment hybridizing to the probe suggesting that the neo τ gene cassette was integrated intact into the YAC. Figure 3 shows the induction of HLA-B7 surface antigen in 16-9 cells fused with the YAC-JS2. The HLA- B7 antigen was detected by treatment of cells with mouse anti-HLA monoclonal antibody (W6/32) followed by treatment with FITC-conjugated anti-mouse IgG. The cells were analyzed by cytofluorography. Panels A and B represent 16-9 cells and panels C and D pools of 16-9 cells fused to YAC-JS2, the GART D142H8.neo.18 YAC clone. The thin line indicates cells not treated with IFN and the heavy line indicates treatment with 1,000 units/mL of the indicated Hu-IFN. Panels A and C show treatment with Hu-IFN-αA/D and panels B and D treatment with Hu-IFN-gαmmα. Fluorescence values shown are not linear but represent the fluorescence detection channels (of which there are 256) of the cytofluorograph. The real value of the x axis shown spans approximately three decades on the log scale. Figure 4 illustrates the integration/deletion plasmid and its use. The schematic diagram shows two- point recombination between the integration/deletion plasmid and YAC insert that results in internal deletions. Moreover, genes with known phenotypes contained within the YAC insert can be localized by expressing the deleted YACs in eukaryotic cells. DNA in the vicinity of the insertion can be rescued and identified, then used to isolate the corresponding cDNA clones. Disruption of gene structure and/or function can provide definitive relationships between the cDNA clones isolated and genes into which the integration/deletion plasmid has inserted. Figure 5 illustrates a restriction map of the integration/deletion plasmid. The plasmid contains the antibiotic G418 resistance gene cassette under control of the thymidine kinase promoter and the LYS2 gene as a yeast selectable marker. The Clal site introduced into the BLUR 13 element was used to linearize the plasmid with Alu sequences at each end. This yielded linear DNA that provided a high frequency of recombination with the human-derived YAC insert. Figure 6 illustrates a Southern blot of PFGE from the deleted YACs. Five Lys2+ transformants were chosen to prepare agarose plugs. The plugs were run on PFGE (0.5 x TBE, 35 second pulse, 170 volts, 15 °C). The blot was probed with the radiolabeled neo gene to identify the chromosome into which the plasmid was integrated. GART D142H8.neo.23 (lane 1), neo.24 (lane 2), neo.25 (lane 3), neo.32 (lane 4), and neo.35 (lane 5). Figure 7 illustrates the localization of integrated plasmid in targeted YAC. A) A quarter of a plug was incubated with restriction endonuclease Nøtl as described in "Experimental Procedures" followed by pulsed-field gel electrophoresis (0.5 x TBE, 35 second pulse, 170 volts, 15°C). The Southern blot was hybridized with three probes separately: a) neo, b) centric vector arm (2.7 kb PvuII and BamHI cut of pBR322), and c) acentric vector arm (1.7 kb PvuII and 2-αmHI cut of pBR322). GART D142H8.neo.l (lane 1), neo.16 (lane 2), neo.17 (lane 3), neo.29 (lane 4), neo.122 (lane 5), and neo.125 (lane 6). B) Schematic diagram showing where the integrated plasmid is located in terms of the two Nøtl sites. Figure 8 illustrates the use of deleted YAC to identify specific genes and cDΝA clones. The schematic diagram outlines how the specific gene or its cDΝA can be isolated with the deleted YAC. YAC D142H8.neo.31 and neo.122 have 50 kb and 20 kb deletions, respectively, and share the targeted site which contains the neo gene. A 16 kb Xhol fragment containing the neo gene was rescued as described in "Experimental Procedures." The 600 bp Hindlll fragment around the Sail site of the rescued DΝA was used to isolate cosmid clone GC1-6. Then a 3 kb EcøRI-C/αl fragment from the cosmid insert was utilized to isolate the corresponding cDΝA, 4pll. E: EcøRI, Ν: Nøtl, S: Sai . Figure 9 illustrates an agarose gel electrophoresis of DΝA and Southern blot analysis. A) Yeast chromosomal DΝAs from random internally-deleted YAC clones were cut with restriction endonuclease EcoRI, run on a 0.8% agarose gel, and blotted onto a nylon membrane. The blot was probed with the radiolabeled cDΝA, 4pll, derived from the Nøtl site close to the acentric vector arm (Figure 8). B) The blot was reprobed with the acentric vector arm. GART D142H8 (lane 1), GART D142H8.neo. l (lane 2), neo.4 (lane 3), neo.5 (lane 4), neo.31 (lane 5), neo.35 (lane 6), neo.51 (lane 7), neo.83 (lane 8), neo.100 (lane 9), neo.104 (lane 10) and neo.l22 (lane ll). Figure 10 illustrates a schematic map of fragmentation vectors pSEl and pSE2, and the procedure used for fragmentation of the parental GARTD142H8.ura.2 YAC. TEL = telomere; CEΝ = centromere. Size of _3αmHI-J3αmHI fragment is 1.7 kb. The BamHI-EcόSI fragment containing the Alu sequence is 8.25 kb; the second i-αmHI-Et'øRI fragment is 9.3 kb. The Nøtl and SacI sites are unique and may be used to rescue the right end of the YAC. Figure 11 shows a Southern blot of YACs to demonstrate the presence of the unique pGC8.10Ε6 sequence. YACs were analyzed by pulsed-field gel electrophoresis. After blotting to nitrocellulose, the filters were hybridized under standard conditions to either a 32 P-labeled 3.0 kb EcoRI fragment of 32 P- labeled pGC8.10E6. The pGC8.10E6 contains 6.0 kb of unique DNA mapping to the leftmost end of GARTD142H8 and was provided by Dr. Robert Donnelly. Filters were washed under stringent conditions. Lane 1 is the B8 YAC. Lanes 2- 10 show YACs obtained after fragmentation. YAC in lane 3 is the same approximate size as B8. Lane 7 illustrates one of the two YACs whose size increased. Variations in hybridization intensity reflect the amount of DNA loaded. Figure 12 YAC J18 and overlapping cosmids. Fragmented YAC J18 (150 kb) which encodes the accessory factor- 1 (AF-1) for the Hu-IFN-gαmmα R was described (70,71). The cosmid libraries were made from the YACs GART D142H8 and J18 as described under "Experimental Procedures" and elsewhere (92). The cosmid libraries were screened with human Cot-1 DNA (BRL) as a probe. The Cot 1 positive cosmids were screened with the TRPl and URA3 gene fragments to identify the cosmids corresponding to the YAC ends. The cosmid GC3-59 and JC8 were identified by hybridization to the TRPl and URA3 gene fragments, respectively. Thus, these cosmids corresponded to the respective ends of YAC J18. Gene walking procedures were then used to identify adjacent cosmids. The DNA fragment (/) without human repetitive sequence derived from one end of the GC3-59 cosmid was used to identify the overlapping cosmids GC8-3 and GC8-10. Similarly the terminal fragment (/) from cosmid GC8-10 was used to identify cosmid JC35. The 14 kb EcøRI subfragment from cosmid GC8-10 (pG8.10E14) was used to identify the transcript from 3xlS polyA + mRNA. Figure 13. Northern blot analysis of RNA isolated from human Raji, HL60 and 3xlS cells. (A) Three micrograms of oligo(dT)-selected mRNA from 3xlS cells was fractionated on a 1.0% agarose gel containing 2.2 M formaldehyde and blotted onto a nylon membrane. The blot was hybridized with the 14 kb EcøRl fragment (pG8.10E14) derived from cosmid GC8-10 preassociated to Cot 10 with sonicated human DNA. A single transcript of 1.8 kb was detected. (B) About one half microgram of oligo(dT)-selected mRNA from Raji and HL60 cells were processed as described above and probed with a 1.8 kb insert from plasmid pJS3. The same size of transcript (1.8 kb) from human cells was detected. Figure 14. Schematic representation and restriction map of the human IFN- gamma Accessory Factor-1 (AF-1) cDNA clones pSKl and pJS3. The dark stippled box indicates the location of the putative coding regions for the signal peptide (SP); the black dark crosshatched box, the transmembrane region (TM); the light stippled boxes, 3' and 5' untranslated regions (3'UT and 5'UT); the extracellular (EC) and intracellular (IC) domains as shown. The restriction endonuclease recognition sites are shown on the diagram. Figure 15. Human IFN-gαmmα accessory factor-1 (AF-1) cDNA (pSKland pJS3) nucleotide and predicted amino acid sequences. Figure 15 A shows the sequence of the cDNA insert in plasmid pSKl. Hydrophobic putative leader and transmembrane regions are highlighted by rectangular boxes. Sites of putative asparagine-linked glycosylation are highlighted by heavy overlining of the amino acids involved. The molecular weight of the putative receptor polypeptide from amino acids 28-337 (310 residues) without consideration of post-translational modification is 35,034; from amino acids 1 to 337, the molecular weight is 37,834. Figure 15B shows the sequence of the cDNA insert in plasmid pJS3. Details are similar to those described for Figure 15 A. The amino acid sequence differs from that in pSKl by a single amino acid substitution: Arg 64 (pSKl) Gln64 (pJS3). Figure 16. Hydropathy plot of the predicted human IFN-gamma accessory factor-1 amino acid sequence. Positive values indicate increasing hydrophobicity. The hydropathic values represent an average obtained from 13 amino acids. Figure 17. Induction of HLA-B7 surface antigen. A. The HLA-B7 antigen was detected by treatment of cells with mouse anti-HLA monoclonal antibody (W6/32) followed by treatment with FITC-conjugated anti-mouse IgG. The cells were analyzed by cytofluorography. Panels A and B represent 16-9 cells and panels C and D pools of 16-9 cells transfected with plasmid pJS3. The thin line indicates cells not treated with IFN and the heavy line indicates treatment with 1,000 units/mL of Hu-IFN-α A/D or 100 units/ ml of Hu-IFN-gαmmα. Panels A and C show treatment with Hu-IFN-α A/D and panels B and D treatment with Hu-IFN- gamma. Fluorescence values shown are not linear but represent the fluorescence detection channels (of which there are 256) of the cytofluorogph. The real value of the x axis shown spans approximately three decades on the log scale. B. This represents the results with 16-9 cells transfected with plasmid pJS4 instead of pJS3, but otherwise the experiment was similar to that of part A. C. This represents the results with 16-9 cells transfected with plasmid pSKl instead of pJS3, but otherwise the experiment was similar to that of part A, panels C and D, except that 500 units/mL of Hu-IFN-g mα were used (Panel B). These experiments with plasmid pSKl were performed with a population of transfected cells that were selected with a fluorescence-activated cell sorter for high class I MHC induction in response to Hu-IFN-g mmα. Figure 18. Amino acid sequence alignment of human AF-1 and some members of the class 2 cytokine receptor family. Alignments were made by the algorithm of Needleman and Wunsch (78) with a gap weight of 5.0 and length weight of 0.3 for the parameters used in the computations. The single extracellular domains of the human AF-1, human (23) and mouse (24) IFN-gamma receptors, the human CRF2-4 (90) and the N- and C-terminal repeated domains of the human (87), mouse (86), and bovine (88,89) IFN-gαmmα receptors are aligned. The following are the abbreviations of the sequences aligned with the AF-1 protein (Hu- IFN-gamma R AF-1): Hu-IFN-gαmmα R (23), Mu-IFN-gαmmα R (24), CRF2-4 (90), Hu-IFN-c.R(N) and Hu-IFN-αR(C) (87), Mu-IFN-αR(N) and Mu-IFN-αR(C) (86) and Bo-IFN-αR(N) and Bo-IFN-c-R(C) (88,89). The αR(N) and αR(C) designate amino and carboxy terminal domains of the extracellular region, respectively. The highly conserved cysteines, prolines, charged and aromatic amino acids are boxed. The conserved aromatic and hydrophobic clusters are indicated by an asterisk. The putative signal peptide (SP) and transmembrane domains (TM) are underlined. The conserved cysteine residues in the AF-1, CRF2- 4 and IFN-gamma Rs are indicated by an arrow over the box of the third alignment section; conserved cysteines in CRF2-4 and IFN-gamma Rs, but not AF-1 are indicated by an arrow over the box of the second alignment section. Figure 19. The accessory factor and interferon α/β receptor region of Chromosome 21q. The IFN-α/β receptor gene and the CRF2-4 gene have been localized to one end of the IFN-α receptor YAC. The GART gene is located in the position indicated on the GART YAC. Dotted lines indicate region of possible overlap between the GART YAC and the IFN-α receptor YAC. The orientation of the GART YAC is known to be as shown, with the centromere to the left and the acentric end toward the telomere of Chromosome 21q. The orientation of the IFN- c. receptor YAC relative to the GART YAC has not been determined; however, the centromere is to the left and proximal to the IFN-α receptor gene. A = Ascl; B = BssH II; N = NøΛ; S = Sfil. Figure 20. Schematic illustration of YACs used to transform 16-9 cells. The phenotype of all the yeast strains containing the YACS shown is Ura + , Trp + , neo + . Hybridization to a probe (- 2 P-labelled by random priming) specific to the left (centric) end of the GART YAC (ρGC8.10E6) indicated that all YACs shown contained an intact centric end. Fusion of yeast containing YACs to 16-9 cells was performed as described under "Experimental Procedures." A " + ' indicates that MHC class I induction with IFN-gamma was observed; *" -* indicates no observed induction. Sizes of YACs were determined by pulsed-field gel electrophoresis (PFGE) with yeast DNA PFGE markers. Small circles represent telomeres; ovals indicate location of centromeres. Figure 21. Cytofluorographic analysis of MHC class I induction in cells transformed by yeast containing Ura+ YACs. Hu-IFN-gαmmα was present at 125 units/ml for 48 hrs in all cases. Solid line, no interferon; stippled area, cells treated with Hu-IFN-gαmmα. Double peaks were observed in the J16/16-9 cell line. This phenomenon may be attributable to loss of the Hu-IFN-gαmmα R or the HLA-B7 gene. In the case of J29/16-9 cells, the original transformants also displayed two peaks but subsequent cloning produced the J29/16-9 cell line exhibiting a single peak after IFN-gamma induction. Figure 22. Responsiveness of cell lines to various concentrations of Hu-IFN-gαmmα. Cells were incubated with or without Hu-IFN-gαmmα at the indicated concentrations for 48 hrs. In each section, points to the left are controls incubated without Hu-IFN-gαmmα. All fluorescence values are based upon the mean fluorescence of the cell population (N = 10,000). Figure 23. Gel shift assay for ISGF3-gαmmα(A) and GAF(B) in selected cell lines. For ISGF3-gαmmα, cells were treated with IFN-gamma at 100 units/ml for 18 hours and then assayed for ISGF3-gαmmα as described under "Experimental Procedures" with an oligonucleotide corresponding to the ISRE (114). For GAF, cells were treated with IFN-gamma at 100 units/ml for 30 minutes and assayed as described ( ). C = control; / = IFN-gamma treated. GART refers to the cell line which was transformed by the parental GART YAC. Figure 24. Diagrammatic representation of the location and interaction of the Hu-IFN-gαmmα receptor accessory factors located on Chromosome 21q and elsewhere. AF-1 refers to the Chromosome 21 accessory factor whose activity and location have been previously described (97,98,101,107,117). AF-2 is an accessory factor which is also located on Chromosome 21q and which confers EMCV resistance to cells in response to Hu- IFN-gαmmα. AF-3 is an accessory factor which is required for VSV resistance (110) in response to Hu-IFN-gαmmα, but not located on Chromosome 21q. Dashed arrows indicate potential cooperative interactions between accessory factors. RESULTS AND DISCUSSION YAC Clone Encoding an Accessory Factor 3xlS Region YACs The 3xlS somatic cell hybrid contains DNA that encodes one component of the human type I interferon receptor (22,43-45) as well as the accessory factors responsible for class I HLA antigen induction upon binding of Hu-IFN-gαmmα to its receptor (14,15,22,46). The segment of human Chromosome 21 in 3xlS cells is referred to as the 3x lS region, whose size is about 1-3 Mb depending on the estimation method (46; R.J. Donnelly, unpublished data). In order to identify the gene(s) for accessory factor(s) for the Hu-IFN-gαmmα receptor system, Jung (46) prepared a cosmid library from 3xlS DNA and screened the library with labeled total human DNA so that cosmid clones containing highly repetitive human sequences (Alu, Kpn etc.) could be selected. More than 200 cosmid clones were isolated which theoretically covered the 3xlS region 2 to 6 times. The resultant cosmids were transfected into CHO cells which had been stably transfected with both the HLA-B7 gene and Hu-IFN-gαmmα receptor cDNA in an attempt to reconstitute a biologically active Hu-IFN-gαmmα receptor system capable of inducing human class I HLA antigen expression. This approach was not successful, possibly because this region may contain several genes necessary for activity which cannot be cloned into a single cosmid, the gene is too large to be accommodated in a single cosmid vector, or there was a bias during screening with human repetitive DNA so that the particular region of DNA was not represented in the cosmid clones positive for human DNA. For these reasons, YAC clones were chosen to carry out a similar experiment to that described above since YAC clones can maintain more than 1 Mb of DNA as an exogenous fragment (27,47). Through the Chromosome 21 Joint YAC Screening Effort, 10 YAC clones were obtained screened with various primer pairs derived from six different loci which are scattered in the vicinity of the 3xlS region. As shown in Figure 1 and Table I, there were four YAC clones [525 B119G7 (1200 kb), 525 A165D7 (210 kb), 525 C4C10 (220 kb), and 525 C14B2 (450 kb)] derived from the D21S65 locus; one YAC clone [H8(517) A222A12 (220 kb)] from the D21S17 locus; one YAC clone [GART D142H8 (540 kb)] from the D21PFGS locus; two YAC clones [518 A234B10 (210 kb) and 518 B134B9 (225 kb)] from the D21S55 locus; one YAC clone [SOD D112A5 (180 kb, 120 kb)] from the D21SOD1 locus; and one YAC clone [IFNAR B49FI (150 kb)] from the D21IFNAR locus (Table I). The SOD, D21S58, GART, D21S65, H8, and IFNAR loci were known to be in the 3xlS region (22). Although the D21S55 locus was not in the 3xlS region, it was close enough to the 3xlS region to be tested. The YAC derived from the D21S58 locus was not available in time to be included in this study. The 525 B119G7 YAC and IFNAR B49F1 YAC are chimeric YACs which have non-contiguous human DNA fragments (Dr. David Patterson, personal communication). The SOD D112A5 YAC clone contains two different YACs, yet it is not known which one is the SOD YAC. Until it is known how the clones overlap, it is difficult to estimate how much of the 3xlS region is covered by these YAC clones. However, if it is assumed that there is no overlap between the YACs from different loci, except the chimeric 525 B119G7 and IFNAR B49FI YACs, then 5 different YAC clones (525 C14B2, 517 A222A12, GART D142H8, 518 B134B9, SOD D112A5) may cover about 1.5 mb of the 3xlS region, which represents at least 50% of the region. Integration of the Neø r Gene into YACs In order to test the function of these YACs, the neo τ gene was introduced into the YACs by targeting homologous recombination to Alu repeat sequences. A similar procedure was described (33) for S. cerevisiae YPH252. Since all of the 3xlS region YACs were derived from S. cerevisiae AB1380 which has a genotype different from that of S. cerevisiae YPH252, a new integrating plasmid, pJSl, containing a modified human Alu family member BLUR13/C/αI, the yeast LYS2 gene, and the neo τ gene was designed as described under "Experimental Procedures." Digestion of pJSl with Clal produces a linear 10 Kb molecule with two free ends homologous to Alu sequences. The C/αl-linearized plasmid should then target integration to Alu sequences on the YAC by homologous recombination. This event introduces the Lys2 gene (and neo τ gene) into the YAC clones so that Lys2 + transformants can be selected on yeast minimal medium lacking lysine. Twenty Lys2 + transformants were selected from each of ten different YAC clones transformed with integrating plasmid pJSl . To check for integration of the neo τ gene into yeast DΝA, each of the twenty Lys2 transformants was screened by colony hybridization with the neo τ gene as a probe (data not shown). The frequency of a neo τ gene in the Lys2 + transformants varied from 5% to 80%. It seems that in some cases only the LYS2 gene was selectively integrated during recombination. To confirm that the neo τ gene was integrated into the YAC rather than the endogenous yeast chromosomes, two neo τ positive clones from each YAC were selected for analysis by PFGE and hybridization with probes for the neo τ gene (Fig 2A). About 76% of the neø r -positive clones were targeted to the YAC rather than yeast chromosome II which contains a mutated LYS2 gene (data not shown). Since the LYS2 gene in S. cerevisiae AB1380 is almost intact, it is possible that the LYS2 gene in the integrating plasmid was targeted to the endogenous yeast LYS2 sequence in some cases. It is also worth noting that the GART D142H8.neo.18 clone turned out to have a smaller YAC than the original one (Figure 2A). In this case, it is possible that two distant Alu sequences were targeted and the internal DNA fragments between them were lost during recombination (48). To test whether the neo τ gene cassette is integrated into the YAC intact, total yeast chromosomal DNA from targeted YAC clones were digested with J5αmHI and Xhol and the blot was probed with labeled neo τ gene. As shown in Figure 2B, 1.1 kb fragments corresponding to the neo τ gene were apparent indicating that the neo τ gene was targeted without disruption of the coding sequence. In some cases (one out of five), DNA fragments of a different size were detected. Fusion and Cytofluorographic Analysis Spheroplasts from the neo τ gene-containing YAC clones were fused to 16-9 cells. After selection with G418 for two weeks, resistant colonies were obtained from several of the YACs (Table 1). Not all fusions generated G418 resistant colonies possibly due to disruption of the neo τ gene during the targeting process. Colonies from the individual fusions were pooled and analyzed for IFN- dependent class I HLA antigen induction by cytofluorography. The parental 16-9 cells displayed very good class I HLA induction upon treatment with Hu-IFN-αA/D (Figure 3, Panel A) and a slight reduction in HLA expression in response to 1,000 units/mL Hu-IFN-gαmmα (Figure 3, Panel B). The 16-9 cells transformed by fusion with GART D142H8.neo.18 (henceforth called YAC-JS2) also exhibited a good response to Hu-IFN-αA/D (Figure 3, Panel C). In addition, they also showed good HLA induction upon treatment with 1 ,000 units/mL Hu-IFN-gαmmα (Fig 3, Panel D). Treatment of these transformed 16-9 cells with 200 units/mL of Hu- FN-gamma also gave a response similar to that for treatment with 1,000 units/mL (data not shown). The results shown in Figure 3 with 16-9 cells fused to YAC-JS2 were performed on several occasions with pools of clones and with individual clones and gave similar results. It is interesting to note that a colony derived from the GART 142H8.neo.16 YAC was negative for HLA induction by Hu-IFN- gamma. This YAC clone may have a deletion of approximately 10 kb since the apparent YAC size is similar to the original GART YAC even after the integration of the 10 kb plasmid (Figure 2A, lane 2). It is difficult to conclude definitively that the negative response resulted from the deletion of a portion of the gene(s) which encodes the Hu-IFN-gαmmα accessory factor (s) because only one colony, which may not have incorporated the entire YAC (31), was assayed. However, if a sufficient number of neø r -positive YACs are screened which do not respond to Hu- IFN-gαmmα, it may be possible to identify the gene(s) disrupted during integration of the pJSl plasmid. Antiviral Activity of 16-9 Cells Transformed with YAC-JS2 It was reported that hamster/human somatic hybrid 153B7-8 cells (carrying chromosome 21q as its sole human chromosome) transfected with the Hu- IFN-gαmmα receptor cDNA are protected against EMCV infection and display an increase in class I HLA antigen expression upon treatment with Hu-IFN-gαmmα (20,25). Furthermore, since the 3xlS region of Chromosome 21q is sufficient to complement the Hu-IFN-g mmα receptor for class I HLA induction (22,46), we tested whether this region also encodes the accessory factor which confers protection against EMCV infection in response to Hu-IFN-gαmmα. The results indicate that the 3xlS region of chromosome 21q encodes the accessory factors for EMCV antiviral protection (Table 2). However, 16-9 cells transformed with YAC- JS2 (expressing the accessory factors for class I HLA antigen induction) do not show any protection against EMCV. This indicates that there are at least two accessory factors on human Chromosome 21q: one is required for induction of MHC class I antigens and a second is required for protection against EMCV. Although expression of proteins has been demonstrated with YACs containing known genes (30-32), this is the first study which demonstrates a specific biological function from an undefined gene in a YAC expressed in eukaryotic cells. In order to introduce the G418 antibiotic resistance gene into the YACs used in this study, a new integrating plasmid (pJSl) was constructed for S. cerevisiae strain AB1380 which has been widely used for construction of YAC libraries. In conjunction with yeast and eukaryotic cell-fusion techniques, the integrating plasmid was used to introduce the neo r gene into the YACs so that the functional activity of the YACs could be determined. Furthermore, the integrating plasmid can also be used to localize the region of the YAC responsible for functional activity through its ability to produce insertional inactivation of genes and deletions. Recombination with plasmid pJSl can produce deletions of various size within a YAC. We have observed deletions of up to 350 kb in the case of the GART D142H8 YAC (49). This study demonstrated that the Chromosome 21 accessory factor gene necessary for class I HLA antigen induction by Hu-IFN-gαmmα is located in the 540 kb of human DNA in the GART D142H8 YAC. This observation is consistent with data which indicated that the gene for the accessory factor is located in the distal portion of band 21q22.1, near the markers D21S58 and GART (21,45). We previously demonstrated that the accessory factors required for VSV protection are different from those leading to EMCV protection after Hu-IFN- gamma treatment (20). The observation that a small segment of human chromosome 21q (3xlS region) was sufficient to induce class I HLA antigen expression and EMCV protection with Hu-IFN-#αmmα suggested that the same accessory factor was involved in both activities (20). However, the observation that 16-9 cells transformed with YAC-JS2 were not protected against EMCV challenge is evidence that another accessory factor is necessary for this antiviral activity, or that both the antiviral factor and the class I MHC induction factor are required for this antiviral activity. Thus our data indicate that the distinct additional factor responsible for the EMCV antiviral effect present in the 3x1 S region is not included in the GART D142H8 YAC. One might argue that the factor for antiviral activity is in the GART D142H8 YAC but the gene was disrupted due to targeting of the integration plasmid. However, because two separate pools of 16-9 cells fused with different neø r -gene targeted YACs (GARTD142H8.neo.3; and YAC-JS2; Table 1) showed MHC class I induction but no protection against EMCV when treated with Hu-IFN-gαmmα, the argument for disruption of the same gene is less convincing. The identification of a YAC which encodes the species-specific accessory factor for class I HLA induction by Hu-IFN-gαmmα should eventually lead to identification of the cDNA for the protein. This information will help us to understand the interactions between the Hu-IFN-^αmmα receptor and the accessory factors. In order to locate the genes and to find cDNAs for the accessory factors, various approaches such as YAC fragmentation (49), exon- trapping (50), hybrid selection of cDNA with the YAC (51), and direct screening of the cDNA library with the YAC (52) can be carried out. We have mapped the GART D142H8 YAC with some restriction endonucleases (Nøtl and Ascl). The mapping data suggest that the GART D142H8 YAC is similar to yGART2 (600 kb) described by Girke et al. (32) since both YACs have two Nøtl sites and the GART gene is located between two Nøtl sites (170 Kb). Our results are consistent with and extend the results of Chumekov et al. (53) who reported a physical map of human Chromosome 21 constructed with the use of overlapping YACs. Our current efforts are directed at defining the specific gene or genes on the GART D142H8.neo.18 YAC that contribute the Chromosome 21 accessory function required for activity of the human IFN-gamma receptor. This study and our previous studies (20,46) demonstrate that accessory factor functions for class I MHC induction, protection of cells against EMCV infection and protection against VSV infection are mediated by different factors or a different combination of factors. We thus conclude that a family of accessory factors that mediate unique intracellular signals is associated with the IFN-gamma receptor. It is likely that these factors are qualitatively and quantitatively different in various tissues, a hypothesis that explains the individual responses of different cells and tissues to IFN-gαmmα. Λ/w-targeting YAC Deletion Plasmids Integration/Deletion Plasmid Construction The parental plasmid for construction of the integration/deletion plasmid was pMClneo polyA which has an antibiotic G418 resistance gene cassette. The EcøRI site in the BLUR 13 fragment from the pBP47 integrating plasmid (33) was modified to a Clal site so that the integration/deletion plasmid could be linearized with Clal resulting in an Alu sequence at both ends. The LYS2 gene was used in the integration/deletion plasmid to introduce a selectable marker into yeast after transformation. The ADE2 gene had previously been utilized to create an integrating plasmid with a selectable marker (34). However, a single copy of the ADE2 gene was not sufficient to confer adenine prototropy so that about 20 copies of the plasmid were necessary for selection. A partial restriction map of the integration/deletion plasmid pJSl is shown in Figure 5. The plasmid pJSl was transfected into NIH3T3 cells and antibiotic G418-resistant colonies appeared after 14 days of selection. This confirmed the presence of a functional neo r gene. Two-point recombination of the pJSl plasmid into the YAC insert can delete the DNA between two separate Alu integration sites. Since Alu family sequences are known to be distributed at an average of 2 to 10 kb apart (61), deletions of various sizes at random positions can be introduced into the YAC insert as well as simple integration into single Alu sequences (Figure 4). Transformation of Yeast and Integration of Plasmid into the YAC Because linear DNA provides greater recombinogenic activity in yeast than circular DNA, plasmid pJSl was linearized with Clal restriction endonuclease and then introduced into GART YAC D142H8 via a PEG- mediated transformation procedure. Twenty Lys2 + transformants were analyzed by colony hybridization with the neo gene as a probe to determine if this gene was integrated into the YAC clones. Sixteen clones out of the twenty Lys2 + transformants showed the presence of a neo gene fragment (data not shown ). The frequency of introducing the neo gene into the YAC clones along with the LYS2 gene (16/20) is higher than the results of Pachinis et al. (34) where only 1 out of 10 Ade transformants was shown to have a neo gene. However, we found that the frequency varies from clone to clone. Neø-negative, Lys2 + transformants might result from the loss of the neo fragment during recombination and/or from reversion of the chromosomal Lys2 " to Lys2 + . Internally-Deleted YACs One hundred twenty-eight Lys2 + transformants were analyzed to determine the size of the deletion in the YAC. While fifteen clones of one hundred twenty- eight (about 11 %) were shown to have the plasmid integrated into yeast chromosome III which contains the endogenous LYS2 gene, sixty clones of the Lys2 + transformants (about 47%) showed the presence of the neo gene in the YAC when analyzed by Southern hybridization of PFGE gels probed with a radiolabeled neo gene fragment. As shown in Figure 6, YACs D142H8.neo23, neo.24 and neo.25 have less than 20 kb deletions, and YAC D142H8.neo.32 and neo.35 have a deletion of about 350 kb and 50 kb, respectively. The size of the deletion in sixty neo targeted YACs was determined (Table 3). The high number of clones having deletions of 0 to 20 kb (38 out of 60) suggests that the Alu sequences at the ends of the integration/deletion plasmid may have topological constraints which limit the targeted sequences to two relatively close Alu sequences in the YAC insert. In order to determine where the recombination occurred, agarose plugs were made from six clones, and the DΝAs were cut with endonuclease Nøtl and analyzed by Southern hybridization with a neo gene probe or probes derived from the centric or acentric YAC vector arms. As shown in Figure 7, YAC D142H8.neo. l was shown to contain the integration/deletion plasmid in a 230 kb Nøtl fragment close to the acentric vector arm since both the neo and the acentric probe hybridized to the same 230 kb fragment, while the centric probe hybridized to 140 kb Nøtl fragment close to the centric vector arm. The centric vector arm probe hybridized to the 230 kb right Nøtl fragment which contains the integration/deletion plasmid as well as the 140 kb Nøtl fragment because the targeting plasmid has part of the centric vector sequences (ampicillin resistance gene). YACs D142H8.neo.16, neo.29, neo.122, and neo.125 were targeted to the middle 170 kb Nøtl fragment and YAC D142H8.neo.17 to the 140 kb Nøtl fragment close to the centric vector arm (Figure 7B). These data suggest that integrations and deletions occurred into Alu sequences throughout the YAC. A few clones were found to have two YACs, one with an integrated plasmid and one without. In those cases, the integrated YAC has a deletion and is positive for the neo gene. Fusion of Yeast Spheroplasts to Mammalian Cells Since the integration/deletion plasmid introduces the antibiotic G418- resistance selectable marker into the YAC, the functional activity of the deleted YAC can be tested following fusion to appropriate mammalian cells (30,32,31). However, when two-point targeting deletes part of the gene, eliminating its functional activity by disrupting the gene, eukaryotic cells with a negative phenotype will be obtained. Based on the location of the deletions in the YAC clones which have lost a functional activity, the specific gene can be precisely located (Figure 4). Since recipient cells do not take up all of the YAC DNA after spheroplast fusion (32), it is important to assay several antibiotic G418-resistant colonies in order to ascertain that the negative phenotype was derived from a deletion or insertion, not from integration of only a portion of the YAC DNA into the eukaryotic genome. The fusion efficiency ranged from 3 x 10 " ^ to 1 x 10 " ° (G418 resistant colonies/input cells). In some cases, no G418 resistant colonies appeared partly because the neo τ gene was disrupted during the integration process. Isolation of DNA Fragments Adjacent to the Integration Sites After identifying the deleted YAC clones which exhibit a lack of functional activity, localization of the inactivated gene can be carried out by rescuing the DNA fragment adjacent to the targeted site. Before rescuing the DNA fragments it should be determined whether or not a single plasmid was integrated into the YAC. If more than one copy of the plasmid was integrated, it would not be possible to assign the site responsible for functional inactivation unambiguously. Chromosomal DNA from YAC D142H8.neo.122 was digested with restriction endonuclease Xhol, separated on a 0.8 % agarose gel, transferred to a nylon membrane, and probed with a radiolabeled neo gene fragment. One band at approximately 16 kb hybridized to the probe indicating that only one plasmid was integrated. The 16 kb Xhol fragment containing the integration sites was isolated from a plasmid library constructed with DNA from YAC D142H8.neo.122 DNA with a neo gene as the probe. The map of the 16 kb rescued plasmid (pNX16) is shown in Figure 8. The insert DNA contains part of the neo gene and human DNA sequences derived from the vicinity of the Nøtl site close to the acentric vector arm. We also found that YAC D142H8.neo.31 has the same 16 kb Xhol fragment hybridizing to the neo gene (data not shown), suggesting that one of the two targeted Alu sites in these two deleted YAC clones occurred at about the same position. The other targeted Alu site of these YAC clones must be different because YAC D142H8.neo.122 and neo.31 have a 20 kb and a 50 kb deletion, respectively (Figure 8). In order to isolate a large intact piece of DNA encompassing the targeted site, a Hindlll fragment (0.6 kb; free of human repetitive DNA) from the 16 kb rescued DNA was used to screen a cosmid library made from the original GART D142H8 YAC clone. A cosmid clone, GC1-6, was isolated. A 3 kb EcoI -Clal fragment from the insert of cosmid GC1-6 which is free of human repetitive sequences was used in turn to screen a cDNA library made from 3xlS human/hamster irradiation reduced hybrid cells (22). A cDNA clone, 4pl l, was obtained (1.5 kb in size). Thus the rescued plasmid was used to locate the gene and isolate its cDNA (Figure 8). Northern hybridization showed that the mRNA corresponding to the cDNA 4pl l is expressed in 3xlS cells (data not shown). Disruption of a Gene by Deletion In order to confirm that the random deletion event causes a deletion of the specific gene, ten deleted YACs were selected randomly for analysis of disruption of the gene. A Southern blot made from EcøRI-digested yeast DNAs was probed with cDNA 4pll (Figure 9). The intact gene hybridizing to the cDNA contains seven EcøRI fragments (Figure 9, lane 1 ). However, YACs D142H8.neo.5 (lane 4), neo.31 (lane 5), neo.51 (lane 7), and neo.122 (lane 11) exhibited smaller fragments than the original 14 Kb EcøRI fragment. YAC D142H8.neo.35 (lane 6) is missing all seven EcøRI fragments. Although YAC D142H8.neo.83 (lane 8) has a 350 kb deletion, the gene corresponding to the cDNA 4pl l appears to be intact since all seven EcøRI fragments are present. In this report we describe the construction of an integration/deletion plasmid for introduction of the antibiotic G418-resistance gene cassette into YAC inserts while at the same time producing deletions of various sizes during the insertion process. The neo τ gene in the YAC is necessary to select mammalian cells with YACs integrated into the nuclear DNA following fusion between yeast and mammalian cells. The integrating plasmid previously described (34) for S. cerevisiae AB1380 does not have the versatility of our approach because it requires a specific DNA fragment derived from the YAC insert to be used as the targeting sequence, and about 20 copies of the plasmid must be integrated in order to convert the yeast cells from Ade " to Ade . The GART YAC D142H8 used here is similar to the yGART2 clone described (32; 600 kb YAC) since both clones have two Nøtl sites yielding a 170 kb Nøtl fragment that encompasses the GART gene. YAC clones screened from the YAC libraries constructed in S. cerevisiae AB1380 have been used to prepare contigs of this region (53). The plasmid described here can facilitate the characterization of the YACs and the identification of the genes on the basis of their function; and can provide another genetic approach for manipulating and modifying the YAC insert in addition to the integration and fragmentation procedures described previously since it produces random internal deletions of various size. Furthermore, in this study we used a model system to develop a method for locating a functional segment within a YAC by gene disruption and isolating its cDΝA. The cDΝA, 4pl l, isolated with the DΝA fragment adjacent to the targeted site (neo side of the targeted site from YAC D142H8.neo.122 or neo.31) was used to demonstrate the disruption of its gene structure based on the disappearance of the 14 kb iϊtøRI fragment. The combination of structural analysis (DΝA mapping) with functional activity provides definitive procedures to locate genes on YACs and to isolate the cDΝAs involved. Yeast Artificial Chromosome Fragmentation Vectors Of the 27 Ura clones analyzed, 21 were found to contain fragmented YACs of molecular sizes ranging from 80 to 500 Kb (Table 4). Two of the YACs were of higher molecular size than the parental YAC (540 kb) and one YAC was approximatmely equal to 540 kb in size. Three Ura + clones did not contain any discernable YAC, either because of very small YAC size, low copy number, or gene conversion. As is shown in Table 4, the fragmented YACs were mostly 80 to 150 kb in size, but larger YACs were produced as well. To characterize further the YACs produced by this procedure, a 6 kb EcøRI fragment subcloned in plasmid pGC8.10E6 was used as a probe to blot the fragmented YACs. These results indicate that a high percentage (89%) of fragmented YACs contain this segment (Table 4, Figure 11). Since pGC8.10E6 maps to the leftmost 50 kb of the B8 YAC, fragmentation must have occurred from the right end. (Deletion from the left end of the YAC would also be expected to produce acentromeric, Trp ' cells.) Nine of 10 yeast strains containing fragmented neo YACs were fused to CHO cells and produced G418-resistant cell lines. The fragmented YACs produced by this procedure have been mapped with restriction enzymes and have been used to align cosmid clones covering this region of Chromosome 21. The frequency of fragmentation obtained by this procedure is significantly higher than that originally reported (48) and is comparable to the high-efficiency YAC fragmentation vectors more recently described (60,58). Although selection of Ura " mutants is a precondition for use of these vectors, it is anticipated that the Ura3 + selection procedure will be useful in that it is applicable to both AB1380 and YPH252 yeast strains. In addition, incorporation of a neomycin resistance gene into the fragmented Ura3 YACs will enable the localization of functional genes on the parental YAC after fusion of yeast spheroplasts to appropriate mammalian cells. Localization of the Human Interferon-gαmmα Receptor Accessory Factor Gene Gene Mapping Using hamster x human somatic cell hybrids, we determined that accessory factor activity is located on human Chromosome 21q (97,98) and on mouse Chromosome 16 (100). Further cytogenetic evidence indicates that the Chromosome 21 accessory factor gene resides within the 1-3 Mb of Chromosome 21q which is contained in the 3xlS somatic cell hybrid (104,117). In addition, the Chromosome 21 accessory factor gene co-segregates with the IFN-α/β receptor gene and is also located near the GART gene (104,105). These relationships are diagrammed in Figure 19. To map more precisely the location of the human Chromosome 21 accessory factor gene, we obtained a series of YACs specific to Chromosome 21 into which we introduced a neomycin resistance gene in order to determine their biological activity by fusion to mammalian cells (105). These experiments indicated that the gene encoding the Chromosome 21 accessory factor activity is located within a 540 kb YAC which also encodes the GART gene and whose address is D142H8. A second YAC, which is 160 kb in length and may partially overlap the GART YAC (Figure 19), encodes a human IFN-α/β receptor gene ( 118) and is located at address B49F 1 ( 105) . Although the GART YAC is known to be oriented as shown in Figure 19, the orientation of the IFN-α receptor YAC has not been established. In addition, the overlap (if any) between the IFN-α. receptor YAC and the GART YAC (dotted lines, Figure 19) has not been defined. To date, no IFN-α receptor probe has hybridized to the GART YAC (R. Donnelly, J. Soh, S. Pestka, unpublished results). This may indicate that the IFN-α receptor YAC is oriented as shown in Figure 19, or that the IFN-α receptor gene is proximal to the GART YAC but there is a gap between these two YACs. If there is such a gap, it is likely to be less than 50 kb in size. Based on digests of human DNA Cheng et al. (119) have shown that the IFN-gamma receptor gene, the CRF2-4 gene (120) and the GART genes are all contained within a 400 kb Mlul restriction endonuclease fragment. These data are consistent with our map shown in Figure 19. The 3' end of the GART gene has been found to be close to 150 kb from the left (centric) end of the GART YAC. The GART gene itself is 40 kb in length (119); and the IFN-α receptor YAC is 160 kb. These numbers account for 350 kb of the 400 kb Mlul fragment. MHC Induction in Cell Lines Transformed by Fragmented YACs To map more closely the location of the human Chromosome 21 accessory factor gene on the GART YAC and to facilitate its identification, a method was devised to fragment the GART YAC from its acentric end (105,106,113,121). Briefly, a fragmentation vector was constructed which contains a 2-αmHI-EcøRI fragment including an Alu sequence, a neo gene, a URA3 gene and a telomere. Recombination of the Alu sequence with homologous sequences in the parental GART YAC produced truncated YACs which were selected on uracil- deficient plates. DNA was isolated and was characterized by pulsed-field gel electrophoresis. YAC size was generally found to range from 80 to 500 kb (Figure 20). Several YACs were shown to contain the GART gene based upon hybridization to a 32 P-labelled GART gene fragment (obtained from Dr. David Patterson). Virtually all of the fragmented YACs hybridized to pGC8.10E6, a plasmid containing a unique 6.0 kb EcøRI fragment which maps to the 50 kb centric end of the parental GART YAC. Therefore, fragmentation occurred from the acentric end. The phenotype of the yeast transformants was determined to be Uπύ "1" , neo + , Trp + . These transformants were next fused to recipient 16-9 somatic cell hybrids which contained the human IFN-gαmmα receptor as well as the HLA-B7 gene. Antibiotic G418-resistant cells were assayed for Chromosome 21 accessory factor activity as previously described ( 110). As is shown in Figure 20 and Figure 21, cell lines J29/16-9, J28/16-9, J16/16-9 and J18/16-9 were responsive to Hu-IFN-gαmmα as seen by induction of MHC gene expression. However, cell lines J20/16-9 and J6/16-9 did not respond to IFN-gamma. These results indicate that the Chromosome 21 accessory factor gene is located within 150 kb from the centric end of the GART YAC. Restriction mapping of the YACs J20 and J18, both approximately 150 kb in size, indicated that these YACs are virtually identical in size (data not shown). However, two additional fusions of yeast cells containing YAC J20 have not produced IFN-gamma responsive cells. We therefore conclude that YAC J20 contains a small deletion relative to YAC J18 which inactivated the accessory factor gene. Yeast cells containing YAC J6 have been fused twice to 16-9 cells without isolation of any IFN-gamma responsive cells. In addition, one other 80 kb YAC was found to be inactive (data not shown). YACs less than 150 kb in size (i.e., with more than 390 kb of the acentric end deleted) were inactive. YAC J18 is the smallest YAC that retained accessory factor activity. MHC class I induction as a function of IFN-gamma concentration with the cell lines produced by the fusions was examined. As shown in Figure 22, half- maximal induction of MHC class I gene expression was elicited at IFN-gamma concentrations between 1 to 10 units/ml. Significant increases in MHC expression could be detected at 1 unit/ml. These responses are similar to those observed in human cells and hamster x human somatic cell hybrids containing human Chromosomes 21 (or 21q) and 6 (or the cloned Hu-IFN-gαmmα R) (97,98,107,117). Transcriptional Activation Because hamster interferon-induced genes have not been isolated, we next examined the induction of two transcription factors whose sequence is conserved in the hamster. The transcriptional activation of interferon-inducible genes has been shown to be stimulated by transcription factors which accumulate in the cytoplasm and migrate to the nucleus (114,122-124). Levels of transcription factor ISGF3- gamma have been shown to increase in the cytoplasm after IFN-gamma treatment (125). To determine how much of the IFN-gamma pathway is functional in the YAC cell lines, we measured ISGF3-gamma in IFN-gamma treated and untreated cells by gel retardation assays with labeled ISRE (114,122-124). As is shown in Figure 23, ISGF3-gαmmα levels are not induced in J6/16-9 and 16-9 cells treated with IFN-gamma. However, IFN-gamma induced ISGF3-gαmmα in J18/16-9 cells. The reason for the high background in J6/16-9 cells is not clear. The unlabeled ISRE competitor oligonucleotide effectively competed out the ISGF3-gαmmα signal. We also examined the induction of GAF, which is induced much more rapidly than ISGF3-gamma. Incubation of 16-9, J6/16-9 and J18/16-9 cells with IFN-gamma for 30 minutes induced GAF in the J18/16-9 cells but not in 16-9 and J6/16-9 cells. This is consistent with the ISGF3-gamma data. We also tested the cell line transformed by the parental GART YAC and found GAF induction in this case as well. Antiviral Activity The Hu-IFN-gαmmα receptor and Chromosome 21q have been shown to be sufficient for an antiviral (EMCV) response in CHO cells (110). In addition, the 3xlS cell line, containing only 1-3 Mb of Chromosome 21q, is also protected by Hu-IFN-gαmmα when challenged by EMCV (105). To determine whether the receptor and the accessory factor encoded on the YACs are sufficient to produce antiviral effects in CHO 16-9, J18/16-9 and J29/16-9 cells, the cells were challenged with EMCV after treatment with various interferons. Human IFN-β and IFN-αA/D(Bgl), were active in protecting the 16-9, J18/16-9 and J29/16-9 cells against EMCV (Table 7). However, even at >3250 units/ml, Hu-IFN-gamma was unable to induce any protection against EMCV on 16-9 and J18/16-9 lines (Table 7); at > 13,000 units/ml, Hu-IFN-gamma was unable to induce any protection against EMCV on J29/16-9 cells. The 153B7-8 cell line transfected with the Hu- IFN-gamma R was found to have high antiviral activity against EMCV with half- maximal protection at 3 units/ml of IFN-gamma. This value closely agrees with the dose-response data for MHC induction in the 16-9 cell lines containing the YAC clones (Figure 22). These data indicate that anti-EMCV responsiveness must require another human gene which is not present on either the J18 or J29 YAC. This gene is, however, located within the 1-3 Mb of Chromosome 21 since the 3xlS and 153B7-8 cells transfected with the human IFN-gamma receptor cDNA are protected from EMCV infection at similar levels of Hu-IFN-gαmmα (105). The 16- 9 cells containing YACs J18 and J29 exhibited a slight increased sensitivity compared to parental 16-9 cells to both Hu-IFN-αA/D and Hu-IFN-β of about 2- fold and 4- to 6-fold, respectively. The significance of this increased sensitivity remains to be determined. The present study localizes the human Chromosome 21 accessory factor to a position which is within 310 kb of the human IFN-α/β receptor gene. This estimate is derived from the fact that the YAC containing the IFN-α/β receptor gene is 160 kb in size and the J18 fragmented YAC, which encodes accessory factor activity, is 150 kb in size. The relationship between the accessory factor gene and the GART gene has not been established with precision; however, the GART gene is not encoded by J18 (data not shown). Precise localization of the accessory factor gene is not yet possible since we do not have any YACs which are larger than J6 (80 kb) and smaller than J 18 (150 kb). J20, which is also 150 kb in size was negative for MHC induction in several assays. If these data result from the fact that YAC J20 is actually slightly more truncated than YAC J18, the accessory factor gene would have to be bounded by the right end of YAC J18. On the other hand, we cannot exclude that the inactivity of YAC J20 is due to a small internal deletion, undetectable by our gross restriction mapping analyses. We have assayed an additional small YAC which is the same size as J6 (J9, 80 kb). This YAC was also negative. Our present data indicate that the accessory factor gene could be located somewhere between 80 and 150 kb from the left (centric) end of the GART YAC. Rather than examine a number of hamster interferon-induced genes (which have not been isolated) in this study, we examined two transcription factors which have been shown to be induced by IFN-gamma. ISGF3-gamma is a 48 kb protein which is the DNA binding component of ISGF3 ( ). Three other proteins associate with ISGF3-gαmmα to form ISGF3. The other transcription factor, gαmmα-activated factor (or GAF), is rapidly induced by IFN-gamma and binds to a DNA sequence different from that of ISGF3-gαm/..α ( ) ' . We found that there is a correlation between MHC class I inducibility and induction of ISGF3-gαmmα and GAF in the cell lines transformed by the fragmented YACs: the cell lines in which Hu-IFN-gαmmα can induce MHC class I surface antigens are also capable of inducing ISGF3-gαmmα and GAF in response to Hu-IFN-gamma (Figure 23). This indicates that the Chromosome 21 accessory factor is required for ISGF3-gαmmα and GAF induction. The role of the accessory factor in this process may be direct or indirect. The accessory factor may be involved only in ligand binding and/or receptor internalization; alternatively, the accessory factor may interact directly with the interferon stimulated gene factors during signal transduction. The observation that cells J 18/ 16-9 and J29/16-9 are not protected from EMCV infection even in the presence of very high (> 3,000 units/ml) IFN-gamma concentrations indicates that, although the accessory factor and the Hu-IFN-gαmmα R are adequate to induce MHC class I antigens and to activate the ISGF3-gαmmα and GAF pathways, an additional factor is required to generate resistance to EMCV. This factor is clearly not located within the J29 YAC. Examination of the parental GART YAC has also shown that this YAC is not sufficient to produce anti- EMCV activity when expressed in CHO cells (105). As is illustrated in Figure 24, we have designated this Chromosome 21 accessory factor that induces MHC class I antigens and transcription factor ISGF3-gamma as AF-1. The factor which is also encoded on Chromosome 21q and is required for EMCV resistance has been designated AF-2. AF-1 is clearly sufficient for ISGF3-gαmmα and MHC induction in J 18/ 16-9 cells (Figures 20-23). Although AF-2 is required for the antiviral EMCV activity in response to Hu-IFN-g mmα, it may function alone or together with AF-1. This will be clarified when the gene or cDNA for AF-2 is isolated. The accessory factor designated AF-3 was defined in a previous study (110) in which it was shown that CHO cells containing Chromosome 21q (and expressing both AF-1 and AF-2) are incapable of generating full VSV resistance in response to Hu-IFN-gαmmα. AF-3 is therefore located on a chromosome other than 21q. As with AF-2, although AF-3 is required for the antiviral VSV activity in response to Hu-IFN-gαmmα, AF-3 may function alone or together with AF-1 and/or AF-2. As is the case for AF-1, AF-2 and AF-3 are species-specific. The conclusions obtained in this study with fragmented YACs which map to human Chromosome 21 should be compared with those recently reported by Kalina et al. (126) using human-mouse hybrid fibroblasts. These Chromosome 21- containing mouse fibroblasts transfected with Hu-IFN-gαmmα R cDNA were found to induce 2', 5'-oligoadenylate-synthetase, resist EMCV, and induce MHC class I antigens upon IFN-gamma treatment. However, when individual clones were examined it was found that EMCV protection was absent in 67% of the cloned cell lines. Therefore, the genes present on human Chromosome 21 are insufficient to produce EMCV protection and other unidentified genes are postulated to be involved. Our experiments with WA17 (a mouse x human somatic hybrid cell line which is trisomic for human Chromosome 21; reference 127) transfected with the Hu-IFN-gαmmα R failed to show anti-EMCV activity in response to human IFN- gamma (Y. Hibino, T. Mariano, B. Schwartz and S. Pestka, unpublished results). We have not succeeded in expressing the fragmented YACs in murine cells. Hence, we are not able so far to recreate the cellular assay systems employed in the present study in mouse cells. In any case, while the data of Kalina et al. (126) support the hypothesis that other species- specific proteins besides AF-1 are required for anti-EMCV activity, the present study identifies a factor (AF-2) encoded by Chromosome 21 which complements AF-1 to produce EMCV resistance. The species-specific proteins required for anti-EMCV activity in the mouse system cannot be AF-2 since they are not on Chromosome 21. In summary, the fact that a specific chromosomal fragment containing a gene for AF-1, which is functional in CHO cells (as judged by MHC class I induction), is not sufficient for either anti-EMCV or anti-VSV activities demonstrates that other factors are required in order to produce the full spectrum of IFN-gamma activities. Our results define the existence of at least three accessory factors AF-1, AF-2 and AF-3 which are required for functional activity in response to Hu-IFN-gα/nmα and its receptor, including MHC class I ISGF3-gαm/nα and GAF induction, as well as anti-EMCV and anti-VSV activity. The full identification and characterization of these accessory factors will be necessary in order to understand how the IFN-gαmmα R signal transduction mechanism is regulated. cDNA Clone Encoding an Accessory Factor Screening of cDNA Libraries The fragmented YAC clone J18 (150 kb) which was derived from YAC GART D142H8 (540 kb) by chromosomal fragmentation was shown to contain the gene encoding the accessory factor (65,70,71). Overlapping cosmid clones derived from GART D142H8 and J18 YACs were aligned as shown in Figure 12 with a small gap of about 10 kb around the Sfil site proximal to the acentric vector arm. These cosmid clones were transfected into 16-9 cells in order to locate the gene more precisely. However, no transformants responding to Hu- IFN-gαmmα were observed. In order to identify any transcripts encoded from the region of Chromosome 21 encompassed by the J18 YAC, Northern blots of polyA + mRNA from 3xlS cells were probed with five individual cosmid inserts and each EcόSl subfragment of more than 6 kb from these cosmids. Suppression of hybridization to human repetitive sequences was accomplished with an excess of total human placental DNA as described under "Experimental Procedures." As shown in Figure 13, we were able to identify one major transcript whose size is 1.8 kb with the 14 kb EcøRI fragment from cosmid GC8-10. The 14 kb DNA fragment was further digested with restriction endonuclease Hindlll into 6 kb, 5 kb and 3 kb fragments each of which was used to probe the 3xlS polyA mRNA in a Northern blot. All three probes hybridized to the same transcript as did the 14 kb fragment but at different intensities, suggesting that coding sequences are scattered throughout the 14 kb fragment. Since the 5 kb fragment produced the strongest signal in the Northern blot hybridization, this fragment was chosen to screen the cDNA gamma library prepared from polyA + RNA from 3xlS cells (73). Ten positive signals from 250,000 plaques were obtained by screening with the same hybridization conditions as used for Northern hybridization and each was isolated after plaque purification. Clones gamma 33-1 and gamma 42-2 contained the largest inserts and were used in subsequent experiments. Clone gamma 42-2 with a 1.6 kb insert was the longest clone. However, as the insert of this clone did not appear to have the beginning of an open reading frame and was shorter than the 1.8 kb size expected from the transcript, a 300 bp fragment (Sail and Espl) from the 5' end of clone gamma 42-2 was used to screen the Daudi plasmid pCDNAl library. Nine positive clones were identified, purified and characterized. Clones pJS3 and pJS4 were found to have a 1.8 kb insert which is about the same size as the transcript identified. In parallel experiments an oligonucleotide (5' GCTCGAAGGCGTAGAGTGAC 3') homologous to the 5' end of clone 33-1 was synthesized and labeled with [α- 32 P]dATP with terminal deoxynucleotide transferase to a specific activity of 5 x 10 9 cpm/μg. It was used to screen about one million phages from the human M426 cell library (73). Six positive clones were identified and purified. The largest clone, gamma pCEV15-l l, contained a 2.3 kbp insert, was sequenced and evaluated further for biological function. The plasmid rescued from phage gamma pCEV15-l 1 was designated pSKl. Sequencing of Hu-IFN-g mmα AF-1 cDNA Clones Both strands of the entire cDNA insert of plasmid pSKl were sequenced as described under "Experimental Procedures." Since the sequences of the plasmids pJS3 and pJS4 were almost identical to the insert of plasmid pSKl, only one strand of these inserts was sequenced. The restriction maps of the DNA insert in clones pSKl (gamma pCEV15-l 1 ) and pJS3 are shown in Figures. 3A and B, respectively. The sequence of the insert of the Hu-IFN-gαmmα AF-1 clone (pSKl), which represented the largest of the cDNA sequences obtained, is shown in Figure 15 A. Including a poly(A) stretch of 39 residues, the cDNA insert of clone pSKl consists of 2255 bases. The 2255 base-pair nucleotide sequence of the cDNA and the deduced amino acid sequence show an open reading frame of 1011 bases (Figure 15A). The 5' -untranslated region of this clone consisted of 648 bases. The 3' untranslated region extended for an additional 557 bases terminating with a poly(A) tail. A conventional polyadenylation consensus sequence (AATAAA) was found 29 bases upstream of the poly (A) tail. The first initiation codon (ATG) of the largest open reading frame is located 649 bases downstream from the beginning of the cDNA (clone pSKl). This methionine (ATG, position 649) is followed by a sequence that appears to encode a hydrophobic signal peptide with a predicted cleavage site after amino acid 90. The cDNA encodes a protein of 337 amino acids ending with the termination codon TGA at position 1660. In addition to the putative NH2-terminal signal peptide, hydropathy index computation of the translated sequence (Figure 16) reveals a hydrophobic domain towards the carboxy terminus of the molecule (amino acids 248-273) compatible with a transmembrane domain. This region is followed by a sequence of overall basic residues, a feature common to the cytosolic face of the membrane-spanning segments of many proteins including the human and mouse IFN-gαmmα receptors (23,24). This suggests an exoplasmic orientation of the amino terminus and cytoplasmic orientation of the carboxyl terminus characteristic of a type I membrane protein (81). If the leader peptide consists of the first 27 amino acids, the extracellular domain consists of 220 amino acids from positions 28 to 247 (Figure 15A). The intracellular domain is predicted to consist of 64 amino acids from positions 274 to 337, the carboxy terminus (Figure 15A). There are six potential N-linked glycosylation sites, all on the extracellular domain (Figure 15A). The high content of serine and threonine residues (15.1 %) makes extensive O- linked glycosylation a possibility (82,83). The amino acid composition of the complete (pre-protein) and the putative mature Hu-IFN-gαmmα receptor AF-1 molecules are summarized in Table 5. The DNA sequence of the cDNA insert in pJS3 (Figure 15B) was almost identical to that of the insert in pSKl (Figure 15 A). There was virtual identity between the sequences of the coding region except for nucleotide position 839 which was "G" in the pSKl insert and an "A" (nucleotide 109) in the pJS3 insert. This corresponds to a substitution of Arg in pSKl to Gin in pJS3 at amino acid 64 of the AF-1 coding sequence (Figures 4A and 4B). The DNA sequence of the coding region of plasmid pJS4 was identical to the of pJS3. Demonstration of Hu-IFN-gαmmα Receptor Accessory Factor Function of the AF-1 cDNA Plasmids pSKl, pJS3 and pJS4 containing the cDNA for the accessory factor under control of the CMV promoter were expressed in 16-9 cells which express the Hu-IFN-gαmmα/R and the human HLA-B7 antigen (65). The parental 16-9 cells displayed very good class I HLA induction upon treatment with Hu-IFN- A/D (Figure 17A,B, panels A) and a slight negative response to 100 units/ml Hu- IFN-gαmmα (Figure 17A,B, panels B). The 16-9 cells transfected with plasmids pJS3 (Figure 17A, panels C and D), pJS4 (Figure 17B, panels C and D) and pSKl (Figure 17C) also exhibited a good response to Hu-IFN-A/D (Figure 17A,B, panels C; Figure 17C, panel A). In addition, they also showed very good HLA class I antigen induction upon treatment with 100 units/ml Hu-IFN-gαmmα (Figure 17A,B, panels D) or 500 units/ml Hu-IFN-gamma (Figure 17C, panel B). A similar response could be generated by treatment of the transfected cells with as little as 10 units/ml of Hu-EFN-gαmmα (data not shown). The data demonstrate the cDNA clone exhibits the accessory factor- 1 (AF-1) function attributed to a sequence on human Chromosome 21: ability of cells to induce class I MHC antigens in response to Hu-IFN-gαmmα. Antiviral Activity of 16-9 Cells Transfected with Plasmids Expressing AF-1 It was reported that hamster/human somatic hybrid 153B7-8 cells (carrying chromosome 21q as its sole human chromosome) transfected with the Hu-IFN- gamma receptor cDNA are protected against EMCV infection and display an increase in class I HLA antigen expression upon treatment with Hu-IFN-gαmmα (20,25). Furthermore, since the 3xlS region of Chromosome 21q is sufficient to complement the Hu-IFN-g mmα receptor for class I HLA induction (65,22,46), we tested whether this cDNA clone (pJS3) encodes the accessory factor which confers protection against EMCV infection in response to Hu-IFN-gαmmα. Previous results indicated Chromosome 21q encodes the accessory factors for EMCV antiviral protection (20). However, 16-9 cells transformed with YAC-JS2 (65) or YAC J18 (71) that express the gene for the accessory factor for class I HLA antigen induction did not show any protection against EMCV. In accordance with these observations, the 16-9 cells expressing the AF-1 cDNA also did not confer upon the cells the ability to protect cells from EMCV or VSV infection in response to Hu-IFN-gαmmα (data not shown). These results indicate that there are at least two accessory factors on human Chromosome 21q: one is required for induction of MHC class I antigens; a second is required for protection against EMCV. Comparison of Human IFN-gamma Receptor AF-1 to Other Protein Sequences Computer-assisted searches of a sequence data bank (GenBank) demonstrated no significant major sequence homology of the AF-1 cDNA or the protein sequence with known sequences in the data bank. However, a comparison of the AF-1 protein sequence to that of the human and mouse IFN-gαmmα receptors (Hu-IFN-g mα R and Mu-IFN-gαmmα R), the human, mouse and bovine IFN-α receptors (Hu-, Mu- and Bo-IFN-αR) and the human CRF2-4 sequences revealed some overall structural homology of the sequences as previously delineated by Bazan (84,85). Conservation of the position of four cysteines residues is unveiled by the alignment (Figure 18). There is a conserved tyrosine adjacent to the third conserved cysteine. It appears that AF-1 is a member of the class 2 receptor family (84,85). Amino acid comparison with other members of the class 2 receptor family were made: IFN-gamma R, mouse (24), human (23); IFN-αR, mouse (86), human (87), bovine (88,89); and CRF2-4, human (90). Significant homology is seen (20-30%, Table 6) in the extracellular domains, and in the conserved portion of the transmembrane region. The AF-1 protein contains a single D200 domain consisting of D100A and D100B domains each of which is proposed to be organized in two β- sheets with 7 β-strands in each sheet similar to the extracellular domain of other class 2 cytokine receptors (84,85,91). There are a few strongly conserved residues in all members of this family (Figure 18). Two pairs of conserved Cys residues are located at positions that allow for an N-terminal and C-terminal disulfide bridge (84,85). The conservation of two proline residues between the SD100A and SD100B /-sheet domains allows for relative conformational independence of the I- sheets and further supports the suggestion that AF-1 is a member of this receptor family. Positions of clusters of aromatic (W, F, Y) and hydrophobic (A, I, L, V, M) residues are also conserved throughout the extracellular domain. The occurrence of an unmatched Cys residue at position 174 of AF-1 and corresponding Cys residue at positions 174 (mouse) and 167 (human) of the IFN-gαmmα Rs (Figure 18, arrow, third alignment section) might allow for the formation of an intermolecular disulfide bridge before or during IFN-gαmmα binding and transduction of the signal for MHC class I induction. There are also aligned Cys residues at corresponding positions of the IFN-#αmmα/Rs (position 128, mouse; 122, human) and CRF2-4 (position 106) but not AF-1 (Figure 18, arrow, second alignment section) that suggests that CRF2-4 may be another member of the IFN- gamma R complex or accessory factor family. Northern Blot Analysis of AF-1 To determine the size of the human transcript for AF-1, blot hybridization was carried out with the polyA + RNA isolated from Raji, HL60 and 3xlS cells and the labeled human AF-1 cDNA as probe. The probe hybridized to a major transcript of 1.8 kb (Figure 13). Minor transcripts of larger size were observed (2.3 kb and 4 kb). The 1.8 kbp cDNA inserts in plasmids pJS3 and pJS4 correspond to the 1.8 kb transcript. The 2.25 kbp cDNA insert in plasmid pSKl corresponds to the 2.3 kb transcript. The presence of a single major transcript for AF-1 is consistent with it being the product of a single-copy gene. We previously demonstrated that the Chromosome 21 accessory factor gene necessary for class I HLA antigen induction by Hu-IFN-gαmmα is located in a 150 kb region of human DNA (65,71). We now show that a cDNA clone encoded on a gene from the YAC clones previously identified exhibits full functional accessory factor- 1 activity. We reported that the accessory factor required for VSV protection (designated AF-3) is different from that leading to EMCV protection (designated AF-2) after Hu-IFN-gαmmα treatment (20,65). The observation that a small segment of human chromosome 21q (3xlS region) in conjunction with the Hu-IFN- gamma receptor was sufficient to induce class I HLA antigen expression and EMCV protection with Hu-IFN-gαmmα initially suggested that the same accessory factor was involved in both activities (83). However, the observation that 16-9 cells transformed with several YACs (65,71) were not protected against EMCV challenge demonstrated that another accessory factor is necessary for this antiviral activity, or that both the antiviral factor and the class I MHC induction factor are required for this antiviral activity. Our data demonstrated that the distinct additional factor responsible for the EMCV antiviral effect present in the 3xlS region was not included in the YAC clones that exhibited accessory factor- 1 (AF-1) activity (65,71). Similarly, the AF-1 cDNA does not confer EMCV or VSV protection to hamster cells in response to Hu-IFN- 'αmmα. The identification of cDNA clones encoding the AF-1 protein will help us to understand the interactions between the Hu-IFN-#«mmα receptor and the accessory factors. This study and our previous studies (17-17,20,65,46) demonstrate that accessory factor functions for class I MHC induction, protection of cells against EMCV infection and protection against VSV infection are mediated by different factors or a different combination of factors. We thus conclude that a family of accessory factors that mediate unique intracellular signals is associated with the IFN-gamma receptor. It is likely that the expression of these factors are qualitatively and quantitatively different in various tissues, a hypothesis that explains the varied responses of different cells and tissues to IFN-gamma. It is significant that the AF-1 protein exhibits meaningful homology to other members of the class 2 receptor family (84,85) that is represented by IFN-gαmmα and IFN-gamma receptors as well as CRF2-4. Although we do not know the molecular mechanisms by which AF-1 enables Hu-IFN-gαmmα to transmit a signal in combination with the Hu-IFN-gαmrøα R, previous results suggested that the extracellular domain of the Hu-IFN-gamma R interacts with AF-1 (17-19). Since AF-1 is a transmembrane protein, it is likely that the extracellular domains of the two proteins interact. The ultimate intracellular signal that initiates the cytoplasmic events expressed as class I MHC antigen induction, however, must be generated by both Hu-IFN-gαmmα R and AF-1. The molecular signals in response to Hu-IFN- gamma R/AF-2 and Hu-IFN-g mmα R/AF-3 must be different from those in response to Hu-IFN-gαmmα R/AF-1 in order to engender effects not seen with the Hu-IFN-gαmmα R/AF-1 combination alone. We do not know whether AF-1 is required for functional activity of AF-2 and AF-3 at this time. However, the identification and isolation of the AF-1 cDNA will facilitate analysis of these interactions. 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Inventors: COOK JEFFREY R, DONNELY ROBERT J, EMANUEL STUART, KOTENKO SERGUEI, MARIANO THOMAS M, PESTKA SIDNEY, SCHWARTZ BARBARA, SOH JAEMOG, COOK, JEFFREY, R., DONNELY, ROBERT, J., EMANUEL, STUART, KOTENKO, SERGUEI, MARIANO, THOMAS, M., PESTKA, SIDNEY, SCHWARTZ, BARBARA, SOH, JAEMOG
Applicants: UNIV NEW JERSEY MED, UNIVERSITY OF MEDICINE & DENTISTRY OF NEW JERSEY
Application year: 1994
Main classification: A61K38
IPC codes: A61K38, A61K39, A61K48, A61K49, A61P31, A61P35, C07H21, C07K14, C12N15